Human innate lymphoid cell precursors: identification, characterization, applications

ABSTRACT

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCP). Still, how ILCP relate to mature tissue-resident ILCs remains unclear. ILCP that are present in the blood and all tested lymphoid and non-lymphoid human tissues were identified. Human ILCP fail to express the signature transcription factors (TF) and cytokine outputs of mature NK cells and ILCs but are epigenetically poised to do so. Human ILCP robustly generate all ILC subsets in vitro and in vivo. While human ILCP express RAR related orphan receptor C (RORC), circulating ILCP can be found in RORC-deficient patients that retain potential for EOMES+ NK cells, T-BET+ ILC1, GATA-3+ ILC2 and for IL-22+ but not for IL-17A+ ILC3. A model of tissue ILC differentiation (‘ILC-poiesis’) is proposed whereby diverse ILC subsets are generated in situ from ILCP in response to environmental stressors, inflammation and infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Appln. 16/463,655, filed May23, 2019, which is a U.S. Natl. Stage of International Appln.PCT/2017/081041, filed on Nov. 30, 2017, which claims the benefit ofU.S. applications 62/468,550 filed Mar. 08, 2017, and 62/428,310 filedNov. 30, 2016, the contents of each of which are incorporated herein byreference in their entireties for all purposes.

BACKGROUND OF THE INVENTION

Innate lymphoid cells (ILC) are a novel family of lymphoid effectorcells that serve essential roles in the early immune response,consisting of cytotoxic cells (NK cells) and ‘helper-like’ ILCs. Thelater are characterized by expression of interleukin-7 receptor(IL-7Rα/CD127) and categorized into three distinct groups based on theirtranscription factors (TF) and signature cytokines productionsimilarities to T helper (T_(H)) cells. Group 1 ILC (ILC1) expressT-BET/TBX21 and produce T_(H)1-associated cytokines IFN-γ and TNF-α.Group 2 ILC (ILC2) secrete T_(H)2-associated cytokines, IL-5 and IL-13via a GATA-3 and RORα-dependent pathway. Group 3 ILC (ILC3) utilizerelated orphan receptor C (RORC encoding RORyt) to drive production ofthe T_(H)17-associated cytokines, IL-17 and/or IL-22 (Serafini et al.,2015; Spits et al., 2013). These different ILC subsets are found indiverse lymphoid and non-lymphoid tissues, and enriched at mucosal siteswhere they play essential roles in barrier function and innate immunedefense (Artis and Spits, 2015; Eberl et al., 2015).

Diverse human ILC subsets were first identified in secondary lymphoidtissues and subsequently reported at several non-lymphoid tissue sites(intestine, lung, liver, skin) (reviewed in (Juelke and Romagnani,2016)).

Group 1 ILCs can produce type 1 cytokines (e.g., IFNγ and TNF) andcomprise Natural killer (NK) cells and ILC1s (Wikipedia). ILC1s areweakly cytotoxic cells closely related to ILC3s (id.). NK cells arecytotoxic innate effector cells (id.). They are distributed throughoutthe blood, organs, and lymphoid tissue and make up around 15% of theperipheral blood lymphocytes (id.). NK cells play a role in tumorsurveillance and the rapid elimination of virus-infected cells (id.).

Two distinct populations of IFN-γ-producing ILC1 have been described. AT-BET⁺ cell expressing high levels of CD 127 (referred as CD127⁺ ILC1)and CD161 but lacking other specific surface markers has been identifiedin tonsil and inflamed intestine (Bernink et al., 2013). In contrast, anintraepithelial ILC 1 expressing NKp44 and CD103 but not CD127 residesat mucosal sites (Fuchs et al., 2013). Both these ILC1s produce IFN-γ inrespond to IL-12 and can be differentiated from NK cells by minimalEomesodermin (EOMES) expression.

Group 2 ILCs can produce type 2 cytokines (e.g. IL-4, IL-5, IL-9, IL-13)(Wikipedia). ILC2s (also termed natural helper cells, nuocytes, orinnate helper 2 cells) play the crucial role of secreting type 2cytokines in response to helminth infection (id.). They have also beenimplicated in the development of allergic lung inflammation (id.). Theyexpress characteristic surface markers and receptors for chemokines,which are involved in distribution of lymphoid cells to specific organsites (id.). They require IL-7 for their development, which activatestwo transcription factors (both required by these cells)-RORα and GATA3.ILC2s are critical for primary responses to local Th2 antigens in thelung but are dispensable for responses to systemically delivered Th2antigens (id.).

Human GATA-3⁺ ILC2 express the chemoattractant receptor CRTh2, IL-25Rand IL-33R (Mjösberg et al., 2011), are widely distributed (Montaldo etal., 2015) (lung, skin, gut, nasal polyp, adipose tissues) and producetype 2 cytokines IL-5 and IL-13 under a variety of physio-andpathological situations (reviewed in (Kim and Artis, 2015)).

Group 3 ILCs are defined by their capacity to produce cytokines IL-17Aand/or IL-22 (Wikipedia). They comprise ILC3s and lymphoidtissue-inducer (LTi) cells (id.). ILC3s are a lymphoid cell populationthat can produce IL-22 and expresses NKp46 (an NK cell activatingreceptor) (id.). Nevertheless, ILC3s differ from NK cells, as they aredependent on transcription factor RORyt, they lack cytotoxic effectors(perforin, granzymes and death receptors) and they do not produce IFNγor TNF (id.). They are found mainly in mucosal tissues and particularlyin the intestinal tract (id.).

Lymphoid tissue inducer (‘LTi’) cells are a subset of ILCs expressingmolecules required for the development of lymphoid tissue (id.). Theyare essential for development of lymphoid organs during embryogenesisand after birth regulate the architecture of lymphoid tissue (id.). Theyhave also been linked to the maintenance of T cell memory (id.).

Group 3 ILC include fetal lymphoid tissue-inducer (LTi) cells as well asadult lineage⁻ CD127⁺CD117⁺ cells that express the transcription factorRORγt and produce the cytokines IL-17A and/or IL-22 (reviewed in(Montaldo et al., 2015). ILC3 have been identified in fetal mesentericlymph nodes and spleen (Cupedo et al., 2009) and in adult tonsils,intestine, spleen, skin, lung, endometrium and decidua. A subset of ILC3express natural cytotoxicity receptors (NCR, including NKp30, NKp44 andNKp46) and are enriched in IL-22-producing cells (Cella et al., 2009).

Murine mature ILC differentiate from hematopoietic stem cells (HSC) viaa common lymphoid progenitor (CLP) to give rise to diverseID2⁺TCF-1⁺PLZF⁺ ILC precursors (ILCP) in fetal liver (FL) and adult bonemarrow (BM) (Constantinides et al., 2014; Yang et al., 2015). Diverse TFand signaling pathways regulate this process in mice (Serafini et al.,2015); in contrast, human ILC development is less well characterized(reviewed in (Juelke and Romagnani, 2016)). NK precursors (NKP) thatgive rise to cytotoxic CD56⁺ NK cells have been identified in FL, BM,cord blood (CB) and adult tonsil (Renoux et al., 2015), whereascommitted ILC3 precursors (ILC3P) that generate IL22-producing NCR⁺ ILC3in vitro are found in tonsil and intestinal lamina propria but notperipheral blood (PB), thymus or BM (Montaldo et al., 2014). A recentstudy identified tonsillar human ILCP that expresses RORyt and candevelop into mature cytotoxic and helper ILC (Scoville et al., 2016).Interestingly, these human NKP, ILC3P and ILCP were CD34⁺ and enrichedin secondary lymphoid tissues but were rare or absent from thecirculation. It was unclear if such ILCP were developmentally related tomature ILC subsets found in tissues.

Innate lymphoid cells are important in the development of the innateimmune response, and serve an important role in protective immunity andthe regulation of homeostasis and inflammation (Wikipedia).Consequently, their dysregulation can lead to immune pathology such asallergy, bronchial asthma and autoimmune disease (id.). To providesources of ILCs, there exists a need in the art for the development ofcompositions and methods for isolating precursor cells of ILCs. Theinvention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses compositions comprising innate lymphoid cellprecursors (ILCPs), uses of the compositions, and methods of making andusing these compositions.

In various embodiments, the compositions comprise a purified populationof innate lymphoid cell precursors (ILCPs), wherein at least 75%,preferably at least 90%, of the cells in the population have thephenotype CD127+CD117+CD3-CRTh2-, and optionally have the phenotypeCD7+, NKp44-, CD94-, and/or Lin-, and/or optionally CD26+, and/orCD62L+. In some embodiments, at least 75%, preferably at least 90%, ofthe cells in the population have the phenotype CD127+CD117+Lin-CRTh2-(wherein Lin- comprises CD3-), and optionally have the phenotype CD7+,NKp44-, and/or CD94-, and/or optionally CD26+ and/or CD62L+. Preferably,at least 90 % of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD7+ or CD127+CD117+Lin-CRTh2-CD94-, andoptionally CD26+ and/or CD62L+; more preferably at least 99 % or 100 %of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+.

In one embodiment, the invention encompasses a method for making apurified population of innate lymphoid cell precursors (ILCPs)comprising providing a human cell sample, and selecting for cells in thecell sample that have the phenotype CD127+CD117+CD3-CRTh2-, andoptionally have the phenotype CD7+, NKp44-, CD94-, and/or Lin-, and/oroptionally CD26+ and/or CD62L+ to provide a population of cells, whereinat least 75% of the cells in the population have the phenotypeCD127+CD117+CD3-CRTh2-, and optionally have the phenotype CD7+, NKp44-,CD94-, and/or Lin, and/or optionally CD26+ and/or CD62L+. In someembodiments, the method comprises selecting for cells in the cell samplethat have the phenotype CD127+CD117+Lin-CRTh2- (wherein Lin- comprisesCD3-), and optionally have the phenotype CD7+, NKp44-, and/or CD94-,and/or optionally CD26+ and/or CD62L+. In some embodiments of themethod, at least 75% of the cells in the population have the phenotypeCD 127+CD 117+Lin-CRTh2- (wherein Lin- comprises CD3-), and optionallyhave the phenotype CD7+, NKp44-, and/or CD94-, and/or optionally CD26+and/or CD62L+. Preferably, at least 90 % of the cells in the populationhave the phenotype CD127+CD117+Lin-CRTh2-CD7+ orCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+; morepreferably at least 99 % or 100 % of the cells in the population havethe phenotype CD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/orCD62L+.

In one embodiment, the invention encompasses a method for making a celltype selected from ILC1, ILC2, ILC3, and NK cells comprising providing apopulation of innate lymphoid cell precursors (ILCPs), subjecting thecell population to an external stimulus, and detecting an increase in acell type selected from ILC1, ILC2, ILC3, and NK cells.

Preferably, at least 90% of the cells in the population have thephenotype CD127+CD117+CD3-CRTh2-, and optionally have the phenotypeCD7+, NKp44-, CD94-, and/or Lin-, and/or optionally CD26+ and/or CD62L+.In some embodiments of the method, at least 90% of the cells in thepopulation have the phenotype CD127+CD117+Lin-CRTh2-(wherein Lin-comprises CD3-), and optionally have the phenotype CD7+, NKp44-, and/orCD94-, and/or optionally CD26+ and/or CD62L+. Preferably, at least 90 %of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD7+ or CD127+CD117+Lin-CRTh2-CD94-, andoptionally CD26+ and/or CD62L+; more preferably at least 99 % or 100 %of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+.

In one embodiment, the method is performed in vivo.

In one embodiment, the invention encompasses a method for treatment of ahuman patient comprising administering to the patient a purifiedpopulation of ILCPs, wherein at least 90% of the cells in the populationhave the phenotype CD127+CD117+CD3-CRTh2-, and optionally have thephenotype CD7+, NKp44-, CD94-, and/or Lin-, and/or optionally CD26+and/or CD62L+. In some embodiments of the method, at least 90% of thecells in the population have the phenotypeCD127+CD117+Lin-CRTh2-(wherein Lin- comprises CD3-), and optionally havethe phenotype CD7+, NKp44-, and/or CD94-, and/or optionally CD26+ and/orCD62L+. Preferably, at least 90 % of the cells in the population havethe phenotype CD127+CD117+Lin-CRTh2-CD7+ or CD127+CD117+Lin-CRTh2-CD94-,and optionally CD26+ and/or CD62L+; more preferably at least 99 % or 100% of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+.

In one embodiment, the invention encompasses a method for screening forcompounds that affect the development of ILCs comprising providing apopulation of innate lymphoid cell precursors (ILCPs), contacting thecell population with a test compound, and detecting a change in thephenotypes of the cells in the cell population.

In one embodiment, the test compound causes a reduction in thedifferentiation of the ILCPs. In one embodiment, the test compoundcauses an increase in the differentiation of the ILCPs.

In one embodiment, the method comprises infusing a mouse with thepopulation of innate lymphoid cell precursors (ILCPs) and administeringthe test compound to the mouse.

In various embodiments, the cells expand without plasticity.

In one embodiment, the invention encompasses a method for expandingILCPs, comprising culturing the purified population of ILCPs accordingto the invention in a culture medium comprises IL-1β and IL-2.

In various embodiments, ILCPs are cultured in a culture medium comprisesIL-1β and IL-2.

In one embodiment, the invention encompasses a method for expanding ILC3cells with minimal plasticity, wherein the culture medium comprisesIL-1β, IL-2 and IL-7.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A to FIG. 1D depict characterization of peripheral bloodCD117⁺ILC. (A) Gating strategy for FACS analysis of human PB ILC. TotalILC were gated on viable CD45⁺ Lin-(CD3⁻ CD4⁻ CD5⁻ TCRαβ⁻ TCRγδ⁻ CD14⁻CD19⁻) CD7⁺ CD127⁺ cells (red). NK cells are identified by CD56^(Dim)(grey), ILC2 are marked by CRTh2⁺ cells (green) and CD117⁺ ILC are gatedon CRTh2⁻ CD117⁺ population (blue). (B) Percentage of total ILC fromviable CD45⁺ and CD117⁺ ILC from total ILC of healthy adult donors inPB. Results from 27 healthy individuals (Median). (C) Expression ofsurface phenotypes (NKp44, IL1R1 and CD69) and intracellulartranscription factor (EOMES, T-BET, GATA-3 and RORyt) profiles of PBCD117⁺ ILC and gut CD117+NKp44^(+/-) ILC. (D) Functional profiles(IFN-γ, IL-13, IL-22 and IL-17A) of PB CD117⁺ ILC and gutCD117⁺NKp44^(+/-) ILC in response to 3 h PMA/iono stimulation. Datarepresentative of at least 3 individuals analyzed from at least 3independent experiments.

FIG. 2A to FIG. 2E depict the transcriptional signature and chromatinlandscape of CD117⁺ ILC. (A) Schematic and gating strategy for CD117⁺ILC and CD34⁺ HSC freshly isolated from peripheral blood of healthydonor by FACS to perform bulk RNA Seq and Chip Seq. (B) Heatmapdepicting normalized ChIPm-Seq signal showed H3K4me2 intensity of 36449high-confidence enhance region that 1989 regions were specificallyidentify from CD117⁺ ILC. Venn diagram showed the three categories ofH3KMe2⁺ region (‘CD34⁺ HSC-specific’; ‘CD117⁺ ILC-specific’ and‘shared’). Regions were considered cell-type specific ifH3K4Me2⁺enrichment levels differed > 2-fold. (C) Heatmap of molecularpathway enrichment for genes located near GREs uniquely identified fromCD117⁺ ILC (blue) and CD34⁺ HSC (orange). (D) Heatmap showing clusteringof 1540 genes differentially expressed by CD117⁺ ILC compared to CD34⁺HSC that identified by RNA seq. (E) Overlap of the 1540 genes highlyexpressed by CD117⁺ ILC from RNA seq and 2283 genes identified from‘CD117⁺ ILC-specific’ H3KMe2⁺ GREs.

FIG. 3A to FIG. 3E depict that cloning reveals multi-ILC lineagepotential of CD117⁺ ILC in vitro. (A) Expansion of bulk cultured CD117⁺ILC (10 days) in stromal cell-free conditions with cytokines (20 ng/mlfor each cytokine). Results from four independent donors; ns, P>0.05;**, P<0.01; ****, P<0.0001 using paired Student’s t test (Median). (B)FACS analysis of bulk cultured CD 117⁺ ILC for surface phenotypes,intracellular EOMES and cytokine expression after 3 h PMA/ionomycinstimulation to identify NK cells (EOMES⁺ cells), ILC1 (IFN-γ⁺ cells),ILC2 (IL-13⁺) and ILC3 (IL-22⁺ and/or IL-17A⁺). (C) Schematic diagramand morphology of CD117⁺ ILC-OP9 stromal co-culture system. (D) SinglePB CD117⁺ ILC were FACS index sorting and cultured on OP9 or OP9-DL4stromal cells for 14-18 days. Cells were stimulated with PMA/iono 3 hbefore analysis for surfaces and cytokines profiles. Positive cloneswere considered when at least 100 viable human CD45⁺ cells were detectedby FACS. Presence of an ILC subset was scored when more than 5% ofcorresponding cytokine was detected in total viable CD45 cells. (E) Piechart depicting all possible ILC combinations detected. Frequency ofeach single or multi ILC differentiation among total positive wells.Data summarized from four independent experiments with one donor each.On average, cloning efficiency was 40% on OP9 and 26% on OP9-DL4 stromalcells.

FIG. 4A to FIG. 4C depict that CD117⁺ ILC effectively gives rise tomulti-ILC lineage in vivo. (A) Schematic diagram of in vivo transferexperiment. (B-C) Newborn BRGS mice were intrahepatic transferred with1-3 X 10⁵ CD117⁺ ILC or CD34⁺ HSC freshly isolated by FACS from PB ofhealthy individuals. The progeny of these populations were analyzed 4weeks post-injection. (B) FACS analysis for lymphocytes and myeloidsurface markers gated on viable human CD45⁺ cells from bone marrow andgut of BRGS mice transferred with CD117⁺ ILC or control CD34⁺ HSC. (C)FACS analysis of different ILC subsets by surface NKp44 and CD117expression, intracellular EOMES and intracellular cytokines (IFN-γ,IL-13, IL-22 and IL-17A) production in lung, gut, liver and spleen ofBRGS mice transferred with CD 117⁺ ILC. Representative data of at least4 mice in each group from 3 independent experiments.

FIG. 5A to FIG. 5E depict that human ILCP accumulate in human immunesystem (HIS) mice. (A) Schematic diagram of generation of HIS mice.1.5-2 X 10⁵ CD117⁺ ILC or CD34⁺ CD38⁻ HSC isolated from human fetalliver were intrahepatic transferred into newborn BRGS mice. Mice wereanalyzed 8 to 9 weeks post-transplantation. (B) Representative FACSanalysis of human ILCP (Lin⁻ CD7⁺ CD127⁺ CD117⁺) in spleen, BM, lung andliver of HIS mice. (C) Percentage of ILCP from total human CD45⁺ inspleen, BM, lung and liver of HIS mice. (Median). (D) FACS analysis ofsurface phenotypes and transcription factors profiles of ILCP and NKcells from spleen of HIS mice. (E) Cytokines production of spleen CD117⁺ILC from HIS mice pre-culture and post-culture on OP9-DL4 with IL-2, -7,-1β, and -23 for 10 days. Cytokines production was analyzed after 3 h ofPMA/iono stimulation. Representative data of 8 mice from at least 3independent experiments.

FIG. 6A to FIG. 6O depict that in vitro bulk and clonal assay of CD117⁺ILC from lymphoid and non-lymphoid organs. Bulk (100-300 cells) orsingle CD117⁺NKp44^(+/-) CD117⁺ ILC from different organs were FACSsorted into 96-well round bottom plate pre-seeded with OP9 or OP9-DL4and supplied with IL-2, -7, -1β and -23 (20 ng/ml each). IntracellularFACS analysis for cytokines production in respond to 3h PMA/ionostimulation was performed to identify ILC1 (IFN-γ⁺), ILC2 (IL-13⁺) andILC3 (IL-22⁺ and/or IL-17A⁺) after 8-10 days bulk culture (A, D, G, J,M) or 14-18 days single cell culture (B-C, E-F, H-I, K-L, N-O).Representative FACS analysis of progeny from bulk CD117⁺ NKp44⁻ ILCisolated from (A) FL, (D) CB, (G) lung, (J) tonsil and CD117⁺ NKp44⁺from (M) tonsil. Pie chart depicting all possible ILC combinations afterclonal expansion of CD117⁺ NKp44⁻ ILC from (B-C) FL, (E-F) CB, (H-I)lung, (K-L) tonsil and (M-N) CD117⁺ NKp44⁺ from tonsil on OP9 andOP9-DL4. See also FIG. 3 legend. Data summarized from at least 2independent experiments with one donor each.

FIG. 7A to FIG. 7C depict the developmental potential of ILCP fromRORC^(-/-) patients (A) FACS analysis of peripheral blood ILC subsetsfrom healthy and RORC^(-/-) patients sample. (B) Percentage of NK^(Dim)and NK^(Br) from viable CD45⁺ cells, ILC, ILC2 and ILCP from total ILCof PB of healthy and RORC^(-/-) patients. Result from 22 healthy donorsand 2 RORC^(-/-) patients. ns, P>0.05; *, P<0.05; **, P<0.01 usingpaired Student’s t test (Median) (C) ILCP from healthy donor orRORC^(-/-) patients were FACS sorted and cultured on OP9-DL4 with IL-2,-7, -1β and -23 for 8 days. Surface phenotypes, intracellular EOMESexpression and cytokines production profiles were analyzed after 3 hstimulation with PMA/iono.

FIG. 8A to FIG. 8F depict flow cytometric analysis of ILCP markers. FACSanalysis of cells from several normal healthy donors was performed usingvarious markers and the percentages of the indicated cells weredetermined.

FIG. 9A to FIG. 9G depict flow cytometric analysis of ILCP markers. FACSanalysis of cells from several normal healthy donors was performed usingvarious markers and the percentages of the indicated cells weredetermined.

FIG. 10 depicts analysis of CD62L and CD26 human ILCP subsets. Gatingstrategy for FACS analysis of lineage depleted human peripheral bloodcells is shown. Within the Lin-CD7+CD127+CD117+ gate (human ILCP) thereis homogeneous expression of CD45RA and variable expression of CD62L andCD26.

FIG. 11 depicts human ILCP clonal expansion. Human ILCP(Lin-CD7+CD127+CD117+) were cloned from indicated tissues after singlecell sorting using OP9 stromal cells (expressing or not DLL4)supplemented with human IL-1b, IL-2, IL-7 and IL-23. Clones were thenanalyzed for cytokine production (IFN-y, IL-13, IL-17A, IL-22) after 3hr stimulation with PMA/ionomycin. Putative ILCP were identified ascytokine non-producers. Absolute numbers of cells in individual ILCPclones are shown.

FIG. 12 depicts human ILCP clone phenotype. Human ILCP clone derivedfrom adult peripheral blood was stained for the indicated cell surfacemarkers.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have characterized the phenotypic, molecular, andfunctional attributes of peripheral blood CD117⁺ ILCs. While cells withthis phenotype were previously proposed to represent human ILC3(Hazenberg and Spits, 2014), it has been unexpectedly discovered thatthese cells are remarkably enriched in multi-potent and uni-potent ILCprecursors (ILCP) that can give rise in vitro and in vivo to all knownILC subsets, including EOMES⁺ NK cells. CD117⁺ ILCP are found not onlyin the circulation, but also in tissues where they retain ILCmultipotency. The identification of systemically distributed ILCPsuggests a model whereby circulating ILCP provide a cellular substratefor ILC differentiation in tissues in response to infection,inflammation, and cell transformation.

In this report, the inventors identify and characterize human ILCprecursors (ILCP) as a subset of Lin-CD7⁺CD127⁺CD117⁺ cells in cord andadult blood as well as fetal liver and several adult tissues. Human ILCPgive rise to all mature ILC subsets that are capable of producing arange of cytokines (IFN-y, IL-13, IL-17A, IL-22) after in vitro cultureor after transfer in vivo to immunodeficient mice. Human ILCP alsogenerate EOMES⁺ NK cells demonstrating their potential for bothcytokine-producing as well as cytotoxic ILCs. This is the first evidencefor a circulating ILCP in any species and further demonstrate the broadsystemic distribution of ILCP within human lymphoid and non-lymphoidtissues including mucosal sites.

Sorting using the markers CD127+CD117+ cells generated about 20% ILCPand 60-80% T cells which are CD3+ (FIGS. 8A or 9A). Excluding Lineagecells (including CD3) strongly enriched ILCP in CD 127+CD 117+ cells(FIGS. 8E-F; FIGS. 9E-G). Exclusion of CRTh2 ILC2 in Lin-CD 127+CD 117+cells provided further ILCP enrichment, generating at least 75% of ILCP(FIGS. 8E-F; FIGS. 9E, 9F and 9G). Sorting using the markers CD 127+CD117+CD7+CRTh2-Lin- generated approximately 90% ILCP (FIGS. 8E and 9F).By excluding CD56+ NK cells (that are the only Lin- cells that expressCD94), a pure population of ILCP could be identified. Accordingly,sorting using the markers CD127+CD117+CRTh2-Lin-CD94- generatedapproximately 100% ILCP (FIGS. 8F and 9G) Within the Lin+ cells, CD3+ Tcells are essentially the only cells that express CD127 or CD117. Assuch, there is essentially no difference in the percentage of ILCPobtained when comparing CD127+ CD117+CRTh2-CD3- versus CD127+CD117+CRTh2-Lin-.

The identification of human ILCP was possible thanks to a robust OP9stromal cell-based assay that could assess ILC potential at the singlecell level. Using this approach, the inventors identified uni-potentILCP that could give rise to IFN-γ⁺ ILC1, IL-13⁺ ILC2 or IL-17A⁺ and/orIL-22⁺ ILC3 as well as multi-potent human ILCP that could generate twoor more ILC subsets. The inventors demonstrate that human CD34⁺ HSCcould develop in vivo into CD117⁺ cells that harbored ILCP withmulti-lineage ILC potential. Taken together, the inventors would proposea model for human ILCP development whereby pluripotent CD34⁺ HSC wouldprogressively differentiate into multi-potent ILCP (with theCD34-CD7⁺CD127⁺CD117⁺CD45RA⁺ phenotype) that can give rise to the threemain ILC groups (including EOMES⁺ NK cells). Both CD34⁺ HSC andmulti-potent ILCP are present in fetal liver suggesting that this tissueis permissive for this transition. It will be interesting to know if theCD 117⁺ ILCP are present in human BM. Previously described humantonsillar ILCP (Scoville et al., 2016) may represent an intermediate inthis pathway. The absence of CD34⁺CD117⁺CD45RA⁺ ILCP in BM, as well asadult and cord blood (Scoville et al., 2016) suggests that these ILCParise locally. The circulating and tissue-resident human ILCP that theinventors describe herein also harbor cells with more restricteduni-potent ILC. While the inventors have not identified a marker thatallows distinction between multi-potent and uni-potent ILCP, theinventors assume that they retain a precursor-product relationship.

Transcriptomic and epigenomic analysis of circulating human ILCPrevealed a signature consistent with a partial specification to the ILClineage. TFs known to be critical for ILC development in mice (includingTCF7, TOX, ID2 and GATA3; (Klose et al., 2014; Seehus et al., 2015; Yagiet al., 2014; Yang et al., 2015)) were clearly up-regulated in ILCPcompared to circulating HSC. In contrast, signatures of early B and Tlymphopoiesis were not obvious, consistent with the inability of thesecells to adaptive lymphocytes in vitro or in vivo. ILC group-definingTFs (BCL2, TBX21, EOMES, RORC) were either absent or expressed at lowlevels suggesting commitment to ILC1, ILC2 or ILC3 was not yetcompleted. Interestingly, the loci encoding these factors were still‘poised’ as evidenced by abundant H3K4Me2 modifications. This chromatinlandscape likely facilitates rapid generation of differentiated ILCsubsets following cytokine-driven expansion (Zook et al., 2016) andcontrasts with the situation in naïve T cells where signature cytokineand TF loci remain inactive with dominant H3K27 methylation (Koues etal., 2016; Shih et al., 2016).

While uni-potent and multi-potent ILCP were identified in every humantissue sample tested, there were clearly differences in the relativeproportions of ILCP that were uni- or multi-potent. It is thereforelikely that each tissue harbors a unique ILCP ‘repertoire’ conditionedby environmental signals. These may include the same growth factors andcytokines that regulate later stages of ILC differentiation (reviewed in(Diefenbach et al., 2014)), that would act on ILCP to induce developmentof a particular ILC subset. Alternatively, stochastic expression ofcytokine receptors may provide a fraction of ILCP with the ability tofurther differentiate. A better understanding of the mechanisms thatregulate ILCP responsiveness within different tissue environments willbe critical for potential therapeutic applications in human disease.

The inventors’ studies highlight the important role for Notch signals inregulating human ILC differentiation from uni-potent and multi-potentILCP. ILCP from tissues and in blood show a greater multi-potency in thepresence of Notch signals (OP9-DL4 culture system). This may indicate ahigher dependence of multi-potent ILCP for Notch-dependent survival andproliferative signals (Chea et al., 2016b). Alternatively, particularILC subsets may be more Notch-dependent in terms of their homeostasis.In particular, NCR⁺ ILC3 subsets in mice are Notch-dependent (Chea etal., 2016a), although the mechanism of action remains unclear. Theincreased frequency of IL-17A and IL-22-producing cells in OP9-DL4cultures at the bulk and clonal levels may reflect a similar requirementin the human system.

The inventors’ analysis of human fetal liver provides the first evidencefor multi-potent ILCP and ILC3-restricted progenitors during gestation.It was remarkable that other uni-potent ILCP were rarely detected inthis tissue, suggesting that at this stage of fetal development, theliver microenvironment may deliver signals that strongly polarize ILCPtowards ILC3. In the mouse, similar findings have been reported(Cherrier et al., 2012). Notch signals have been proposed to play a rolein directing lymphoid cell fate decisions in the mouse fetal liver,promoting the development of T-lineage primed precursors but alsomodifying homeostasis of ILCP (Chea et al., 2016b; Dallas et al., 2005).Soluble factors are also likely to be involved as ILCP express severalcytokine receptors (IL-1R, IL-2R, IL-18R) that allow them to sensetissue inflammation and stress.

Regulation of TF expression dictates ILC fate as well as function.Signature TF have been identified for ILC subsets that ‘fix’ theirdifferentiation at the level of surface phenotype and effector outputs,especially for cytokines (reviewed in (Serafini et al., 2015)). The TFRORC helps define the ILC3 subset and is required for development andmaintenance of ILC3 (but not ILC1, ILC2 or NK cells) in mice (Luci etal., 2009; Sawa et al., 2010). As expected, RORC is expressed by humanILC3 and in committed ILC3P (Montaldo et al., 2014). The recent reportthat all human ILC subsets express RORC (Scoville et al., 2016)suggested a broader role for this TF in human ILC differentiation. Byanalyzing blood from RORC-deficient patients, the inventors could showthat RORC was not required for global ILC differentiation in humans, butrather was critical for the differentiation of the IL-17⁺ ILC3 subset.ILCP in RORC-deficient patients retained the capacity to generate otherILC and NK cell subsets. Interestingly, IL-22⁺ ILC3 developed in aRORC-independent fashion, suggesting compensatory pathways for thesecells in humans.

The use of OP9 stroma was already shown to minimize human ILC2plasticity (Lim et al., 2016) and here the inventors show that the vastmajority of NKp44+ ILC3 clones retain their functional attributes andshow little plasticity towards the ILC1 phenotype in this culturesystem. Moreover, previous reports proposed that ILC1 clones rapidlydifferentiate towards an ILC3 fate in the presence of IL-1b (Bernink etal., 2015), whereas ILC1 clones in the inventors’ OP9 culture system(containing IL-1β) retained their IFN-γ signature. As such, theinventors’ culture system appears useful to assess signals that promote‘primary’ ILC fate from ILCP.

Finally, the inventors’ identification of circulating andtissue-resident human ILCP suggests a concept of ‘ILC-poiesis on-demand’in which ILC differentiation can occur in any tissue and at any age. Arecent study using parabiosis in mice has proposed that ILCs arelong-lived tissue-resident cells that do not recirculate understeady-state and some inflammatory conditions (Gasteiger et al., 2015).In contrast, other reports have indicated that the half-life of severalmucosal ILC subsets is on the order of weeks, suggesting that thesecells must be renewed (Sawa Science). The discovery of a circulatingILCP provides a mechanism to replenish tissue ILCs in response tosteady-state losses and in the context of infection and inflammation.The invention encompasses compositions comprising and methods of makingand using ILCPs.

Compositions Comprising ILCPs

The invention encompasses compositions comprising innate lymphoid cellprecursors (ILCPs) as described herein. All of the markers used herein(e.g., in the Examples) are specifically contemplated in any and allcombinations for use as markers of ILCPs and can be used in variousembodiments of the invention.

In one embodiment, the invention encompasses a purified population ofinnate lymphoid cell precursors (ILCPs). Preferably, at least 25%, 35%,50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells in the populationhave the phenotype CD34-CD7+CD127+CD117+CD45RA+. Preferably, at least25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells in thepopulation lack the expression of NKp44 and/or RORyt. Preferably, atleast 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cellsin the population are IL-1R1+ and/or CD69-. More preferably, at least25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells in thepopulation have the phenotype CD34-CD7+CD127+CD117+CD45RA+, lack theexpression of NKp44 and/or RORyt, and are IL-1R1+ and/or CD69-. Morepreferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population have the phenotypeLin-CD34-CD7+CD127+CD117+CD45RA+, lack the expression of NKp44 and/orRORyt, and are IL-1R1+ and/or CD69-, and optionally further expressCD62L and/or CD26.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population are Lin-CD7+CD127+CD117+.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD7+CD127+CD117+CRTh2-.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population are Lin-CD94-CRTh2-CD127+CD117+.

Most preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%,or 99% of the cells in the population are CD127+CD117+CD3-CRTh2-. Insome embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%,or 99% of the cells in said population are further CD7+, NKp44-, CD94-,and/or Lin-, and/or further CD26+, and/or CD62L+. In some embodiments ofthe method, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population are CD127+CD117+Lin-CRTh2- (wherein Lin-comprises CD3-), and optionally further CD7+, NKp44-, and/or CD94-,and/or optionally further CD26+ and/or CD62L+. Preferably, at least 75%,80%, 85%, 90%, 95%, or 99% of the cells in the population areCD127+CD117+Lin-CRTh2- (wherein Lin- comprises CD3-), and optionallyfurther CD7+, NKp44-, and/or CD94-, and/or optionally further CD26+and/or CD62L+. In some preferred embodiments, at least 90 % of the cellsin the population have the phenotype CD127+CD117+Lin-CRTh2-CD7+ orCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+; morepreferably at least 99 % or 100 % of the cells in the population havethe phenotype CD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/orCD62L+.Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%,or 99% of the cells in the population do not produce IL-17A or IL-22after stimulation under conditions that these cytokines are produced bygut CD 117⁺ cells.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population do not express T-BET, EOMES, andGATA-3^(hi).

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population do not express CD94, CD244, and CRTh2.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population do not produce IL-13 or IFN-γ afterstimulation with pharmacological activators.

Preferably, the population of cells comprises at least 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, or 10¹⁰ ILCPs cells.

Methods for Making ILCPs

The invention encompasses methods for making a purified population ofinnate lymphoid cell precursors (ILCPs) of the invention. In oneembodiment, the method comprises providing a human cell sample, andselecting for ILCPs in the sample. The ILCPs can be selected with anycombinations of the markers set forth herein (e.g. Examples). In oneembodiment, the cells are selected for any combination of the followingmarkers Lin-, CD34-CD7+CD127+CD117+CD45RA+, NKp44-, RORyt-, IL-1R1+,CD69-, CD62L+, CD26+; in particular, Lin-, CD34-CD7+CD127+CD117+CD45RA+,NKp44-, IL-1R1+, CD69-, CD62L+, CD26+. Lin- refers to lineage negativecells; Lin- includes and refers to CD3⁻, CD4⁻, CD5⁻, TCRαβ⁻, TCRαβ⁻,CD14- and CD19-. Selection can be performed by routine techniques inart, such as by FACS analysis and cell sorting, for example, asdescribed in the Examples.

In some embodiments, the cells are selected for lack of production ofIL-17A or IL-22 after stimulation under conditions that these cytokinesare produced by gut CD117⁺ cells.

In some embodiments, the cells are selected for lack of expression ofT-BET, EOMES, and GATA-3hi.

In some embodiments, the cells are selected for lack of expression ofCD94, CD244, and CRTh2.

In some embodiments, the cells are selected for lack of production ofIL-13 or IFN-γ after stimulation with pharmacological activators, forexample as described in the Examples.

In some embodiments, the sample is a blood or tissue sample. The samplecan be an adult or fetal sample. In some embodiments, the sample is ablood, tonsil, gut, fetal liver, or lung sample.

In some embodiments, the cell sample is selected forLin-CD7+CD127+CD117+CRTh2-cells, such as by cell sorting. In someembodiments, the cell sample is selected for Lin-CD94-CRTh2-CD127+CD117+cells. In some embodiments, the cell sample is selected forCD127+CD117+CD3-CRTh2- cells. In some embodiments, the cell sample isselected for CD127+CD117+CD3-CRTh2-cells and further for CD7+, NKp44-,CD94-, and/or Lin- cells, and/or further for CD26+ and/or CD62L+ cells.In some preferred embodiments, the cell sample is selected forCD127+CD117+Lin-CRTh2- cells (wherein Lin- comprises CD3-), andoptionally further for CD7+, NKp44-, and/or CD94- cells, and/oroptionally further for CD26+ and/or CD62L+ cells. Preferably, the cellsample is selected for CD127+CD117+Lin-CRTh2-CD7+ orCD127+CD117+Lin-CRTh2-CD94- cells, and optionally further for CD26+and/or CD62L+ cells; more preferably, the sample is selected forCD127+CD117+Lin-CRTh2-CD94- cells, and optionally further for CD26+and/or CD62L+ cells. Routine techniques in the art, such as those setforth in the examples, can be used to select these cells. In preferredembodiments, cells are sorted using well-known methods in the art. Insome embodiments, FACS or MACS® Technology (Miltenyi Biotech) are usedto isolate particular cell types. Antibodies against any of the markersdescribed herein can be used to achieve isolation, purification, and/ordetection of any of the cell markers described herein.

Methods for Expanding ILCP

The invention encompasses methods for ILCP proliferation be added. Arobust proliferation of the cells is observed when they are culturedwith IL-1β. For the proliferation of ILCP, the culture medium comprisesIL-1β and preferably IL-1β and IL-2. Furthermore, the medium canoptionally comprises other cytokines, such as IL-7.

The cells can be grown as set forth in the examples, (e.g., Example 3)or by other similar techniques. For example, ILC can be cultured inYssel’s medium with Human AB serum, Stromal cells, IL-7, IL-2, and IL-1βcan be used. Alternatively, other media, such as DMEM, IMDM, orRPMI-1640, can be used. Media and/or media supplements can be varied asknown in the art for cell culture. Also contemplated is supplementationof cell culture medium with mammalian sera.

The media preferably contains a serum selected from bovine serum, calfserum, fetal calf serum, newborn calf serum, goat serum, horse serum,human serum, chicken serum, porcine serum, sheep serum, rabbit serum,and rat serum, or a serum replacement or embryonic fluid. Additionalsupplements, such as amino acids, can be added to the medium.Antibiotics and antimycotics can also be added to the medium

Methods for Making ILC1, ILC2, ILC3, and NK Cells

The invention encompasses methods for making ILC1, ILC2, ILC3, and NKcells. ILC1, ILC2, ILC3, and NK cells can be produced from the ILCPs ofthe invention by routine techniques in the art. For example, ILC1, ILC2,ILC3, and NK cells can be produced using the specific techniquesdisclosed in the Examples. In one embodiment, a cell system (e.g., theOP9 stromal cell system disclosed in Mohtashami, et al. (2010)) can beused to generate ILC1, ILC2, ILC3, and NK cells from the ILCPs of theinvention. Most preferably, the cells expand without changes inphenotype or function also termed “plasticity.”

In various embodiments, the ILCPs are treated with various cytokines topromote differentiation into ILC1, ILC2, ILC3, and NK cells. Thesecytokines include any and all combinations of IL-1β (IL-1 beta), IL-12,IL-18, IL-25, IL-33, IL-23, IL-2, and IL-7.

In various embodiments, ILC subsets can be expanded using a stromalcell-based approach. While others have also shown that mature ILC can beexpanded in vitro, the inventors’ results are different since in thatcase the cells expand without ‘plasticity’ (change in effector function,especially for cytokine production such as IFN-γ). This plasticity canbe driven by a particular human cytokine (IL-12) as the inventors showedin an earlier publication for ILC2 (Lim et al, J Exp Med 2016). ILC3subsets can be expanded in vitro with minimal plasticity using the sameapproach. This approach can be used to grow large quantities of matureILC2 or ILC3 without changing their functional properties.

ILC subsets can be expanded from ILC subsets generated from isolatedILCPs or from ILC subsets directly isolated from patient samples.

The cells can be grown as set forth in the examples or by other similartechniques. For example, ILC can be cultured in Yssel’s medium withHuman AB serum, Stromal cells, IL-7, IL-2, and IL-1β can be used.Alternatively, other media, such as DMEM, IMDM, or RPMI-1640, can beused. Media and/or media supplements can be varied as known in the artfor cell culture. Also contemplated is supplementation of cell culturemedium with mammalian sera.

The media preferably contains a serum selected from bovine serum, calfserum, fetal calf serum, newborn calf serum, goat serum, horse serum,human serum, chicken serum, porcine serum, sheep serum, rabbit serum,and rat serum, or a serum replacement or embryonic fluid. Additionalsupplements, such as amino acids, can be added to the medium.Antibiotics and antimycotics can also be added to the medium.

Most preferably, a medium containing IL-7, IL-2, and IL-1β is used.

Human ILCPs have been expanded in vitro using cytokines in the absenceof stromal cells. Other cell lines can be used for culturing ILCP.

Preferred cell sources of ILCP are peripheral blood, but can alsoinclude bone marrow, tonsils, lymph nodes, skin, adipose tissue, gut,liver and lung. ILCPs from all of these different tissues can becultured in vitro and give rise to mature ILC subsets.

Particular growth factor combinations can be added to the culture mediumto differentiate the ILCP into a specific subset. For example, IL-12 andIL-1β can be added to generate the ILC1 subset. IL-25 and IL-33 can beadded to generate the ILC2 subset. IL-23 can be added to generate theILC3 subset.

Particular growth factor combinations can be added to the culture mediumto inhibit differentiation of the ILCP into a specific subset. Forexample, small molecules, chemical agents or genetic modifications thatalter Tbet or TBX21 expression can be used to inhibit differentiationinto the ILC1 subset. Small molecules, chemical agents or geneticmodifications that alter BCL11B expression can be used to inhibitdifferentiation into the ILC2 subset. Small molecules, chemical agentsor genetic modifications that alter RORyt (RORC in mouse) expression canbe used to inhibit differentiation into the ILC3 subset.

The OP9 cell line is available through ATCC (open access). OP9 cellshave been used previously to develop early human T cell precursors, forexample in US 8,772,028 and US 9,533,009.

The method for expanding ILC3with minimal plasticity differs from thatdescribed for ILC2 in the Lim et al. J Exp Med 2016. For ILC2, matureILC2 (isolated from blood) were cultured (on OP9) with IL-2, IL-7, IL-25and IL-33. For ILC3, mature ILC3 are cultured with IL-2, IL-7 and IL-1β.(see Example 3 where mature ILC3 are isolated from tonsils and culturedon OP9-DL4 with IL-2, IL-7 and IL-1β)

In addition to tonsils, the procedure to generate ILC3 has beensuccessfully used with fetal liver, cord blood, adult peripheral blood,lung, fat and gut samples.

Either the ILCPs or the ILC1, ILC2, or ILC3 cells could also be modifiedby CRISPR, ZFNs, or TALENs, or other genomic editing technologies to addor eliminate desired genomic sequences. Vectors, including retroviral,AAV, and lentiviral vectors, can also be used to modify these cells. Invarious embodiments, a gene selected from RORC or RORTγt, BCL11B, Tbet,and TBX21 is inactivated.

The cells can also be modified to contain a chimeric antigen receptor(CAR). These CARs typically comprise a single-chain binding domain, suchas from a monoclonal antibody or nanobody, fused to a transmembranedomain and endodomain that results in the transmission of a signal inresponse to binding of the binding domain to its target. Examples ofCARs are well-known in the art. Such a genetically-engineered receptor,can be used to graft the specificity of a monoclonal antibody onto amature ILC. ILCs expressing CARs may be useful in some autoimmunediseases since some subsets of ILCs (e.g. ILC2) suppress immuneresponses through myeloid cells.

In various embodiments, the ILCPs are administered in vivo to promotedifferentiation into ILC1, ILC2, ILC3, and NK cells.

In various embodiments, the method comprises providing a population ofinnate lymphoid cell precursors (ILCPs), subjecting the cell populationto an external stimulus, and detecting an increase in at least one celltype selected from ILC1, ILC2, ILC3, and NK cells. In variousembodiments, the ILC1, ILC2, ILC3, and/or NK cells are separated,purified, and/or harvested.

The cell population can be subjected to an external stimulus in vivo orin vitro. In some embodiments, the cell population is subjected to anexternal stimulus in a humanized mouse model. In various embodiments,the external stimulus is a viral, parasitic, microbial, or bacterialorganism (e.g. HIV or malaria) or a component thereof (e.g., DNA orprotein). In various embodiments, the external stimulus is a cytokine ormixture of cytokines. In various embodiments, the external stimulus is atest compound.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population have the phenotypeCD34-CD7+CD127+CD117+CD45RA+, lack the expression of NKp44 and RORyt,and/or are IL-1R1+ and CD69-, and optionally further express CD62Land/or CD26. Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%,90%, 95%, or 99% of the cells in the population have the phenotypeLin-CD34-CD7+CD127+CD117+CD45RA+, lack the expression ofNKp44 and RORyt,and/or are IL-1R1+ and CD69-, and optionally further express CD62Land/or CD26.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD7+CD127+CD117+CRTh2-. Preferably, at least 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD7+CD127+CD117+CRTh2-; more preferably, at least 90%, 95%, or 99%of the cells in the population are Lin-CD7+CD127+CD117+CRTh2-.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD94-CRTh2-CD127+CD117+. Preferably, at least 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD94-CRTh2-CD127+CD117+; more preferably, at least 90%, 95%, or 99%of the cells in the population are Lin-CD94- CRTh2-CD127+CD117+; stillmore preferably at least 99 % or 100 % of the cells in the populationare Lin-CD94-CRTh2-CD127+CD117+.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population are CD127+CD117+CD3-CRTh2-. Morepreferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population are CD127+CD117+CD3-CRTh2- and furtherCD7+, NKp44-, CD94-, and/or Lin-, and/or further CD26+, and/or CD62L+.In some embodiments of the method, at least 25%, 35%, 50%, 60%, 75%,80%, 85%, 90%, 95%, or 99% of the cells in the population areCD127+CD117+Lin-CRTh2- (wherein Lin- comprises CD3-), and optionallyfurther CD7+, NKp44-, and/or CD94-, and/or optionally further CD26+and/or CD62L+. Preferably, at least 75%, 80%, 85%, 90%, 95%, or 99% ofthe cells in the population are CD127+CD117+Lin-CRTh2- (wherein Lin-comprises CD3-), and optionally further CD7+, NKp44-, and/or CD94-,and/or optionally further CD26+ and/or CD62L+. In some preferredembodiments, at least 90 % of the cells in the population have thephenotype CD127+CD117+Lin-CRTh2-CD7+ or CD127+CD117+Lin-CRTh2-CD94-, andoptionally CD26+ and/or CD62L+; more preferably ar least 99 % or 100 %of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+.

Methods and Compositions for Treatment

The invention encompasses compositions comprising innate lymphoid cellprecursors (ILCPs) for use to treat patients in need of innate immunesystem regulation. Thus, the invention encompasses the use of thesecompounds to treat patients and the methods for treating them.

In various embodiments, the patients have a helminth infection, entericpathogen infection, tumor, viral infection, allergy, asthma,inflammation or autoimmune disease (e.g., multiple sclerosis, systemiclupus erythematosus, or type I diabetes mellitus).

In various embodiments, the patients can be immune deficient,immunocompromised, or immune suppressed. In various embodiments, thepatient is a cancer patient or has a chronic disease (e.g. Crohn’sdisease, IBD).

In various embodiments the invention encompasses a method for treatmentof a human patient comprising administering to the patient a purifiedpopulation of ILCPs, wherein at least 90% of the cells in the populationhave the phenotype CD34-CD7+CD127+CD117+CD45RA+, lack the expression ofNKp44 and RORyt, and/or are IL-1R1+ and CD69-, and/or optionally CD26+,and/or CD62L+.

In various embodiments the invention encompasses a method for treatmentof a human patient comprising administering to the patient a purifiedpopulation of ILCPs, wherein at least 90% of the cells in the populationhave the phenotype Lin-CD34-CD7+CD127+CD117+CD45RA+, lack the expressionof NKp44 and RORyt, and/or are IL-1R1+ and CD69-, and/or optionallyCD26+, and/or CD62L+. In some embodiments, at least 90%, 95%, or 99% ofthe cells in the population are Lin-CD7+CD127+CD117+CRTh2-. In someembodiments, at least 90%, 95%, or 99% of the cells in the populationare Lin-CD94-CRTh2-CD127+CD117+.

Preferably, at least 90%, 95%, or 99% of the cells in the population areCD127+CD117+CD3-CRTh2-. More preferably, at least 90%, 95%, or 99% ofthe cells in the population are CD127+CD117+CD3-CRTh2- and further CD7+,NKp44-, CD94-, and/or Lin, and/or further CD26+ and/or CD62L+. In somepreferred embodiments, at least 90%, 95%, or 99% of the cells in thepopulation are CD127+CD117+Lin-CRTh2- (wherein Lin- comprises CD3-), andoptionally further CD7+, NKp44-, and/or CD94-, and/or optionally furtherCD26+ and/or CD62L+. Preferably, at least 90 % of the cells in thepopulation have the phenotype CD127+CD117+Lin-CRTh2-CD7+ orCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+; morepreferably at least 99 % or 100 % of the cells in the population havethe phenotype CD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/orCD62L+.

In various embodiments, uni-potent and multi-potent ILCP, especiallymulti-potent ILCP, can be combined with appropriate factors to make themdifferentiate in vitro or in vivo, into a specific type (ILC1, ILC2 orILC3), depending on the disease to be treated. In some embodiments, itmay be beneficial to either augment or inhibit differentiation of ILCPor to inhibit differentiation into a specific type, depending on thedisease (See, e.g., WO2016/138590, US2016/0145344, and US2016/0304574,which are hereby incorporated by reference).

ILC subsets are involved in various diseases and cellular processes,including infections, cancer inflammation, tissue repair, andhomeostasis. Tait Wojne et al, 2016, which is incorporated by referenceherein. Since ILC3s promote GALT formation, inflammation, immunity, andhomeostasis in the intestine (id.), ILC3s generated by the methods ofthe invention can be used to treat diseases involving these processes.Since ILC2s influence inflammation, immunity, tissue repair, andhomeostasis through interactions with hematopoietic and nonhematopoieticcells (id.), ILC2s generated by the methods of the invention can be usedto treat diseases involving these processes. Since ILC1s express T-betand IFN-γ and contribute to type 1 inflammation (id.), ILC1s generatedby the methods of the invention can be used to treat diseases involvingthese processes.

The cells can be administered to the patient by routine techniques inthe art. Preferably, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ ILCPs cellsare administered to the patient.

Autologous, allogeneic, or xenogeneic ILCs or ILCPs can be administeredto a subject, preferably a human, by direct injection into a tissue orblood, etc. Preferably, the cells are administered in combination with apharmaceutically acceptable carrier. The cells can be administered in asingle or at least 2, 3, 4, 5, etc. injections. The cells can begenetically modified to alter their immune recognition.

Screening Methods Using ILCPs

The invention encompasses methods for screening for compounds thatmodulate (i.e., inhibit or enhance) the differentiation of ILCPs intoILC1, ILC2, ILC3, and/or NK cells. In various embodiments, a populationof the ILCPs of the invention is contacted in vivo or in vitro with atest compound and the effect of the compound on differentiation isassessed. The effect can be observed by detecting a change in thephenotypes of the cells in the cell population.

The test compound can be a natural compound or a synthetic compound. Invarious embodiments, the test compound is a viral, parasitic, microbial,or bacterial organism (e.g. HIV or malaria) or a component thereof(e.g., DNA or protein). In various embodiments, the test compound is acytokine or mixture of cytokines.

In some embodiments, a change in the phenotypes of the cells in the cellpopulation is detected by measuring the levels of ILCPs, ILC1, ILC2,ILC3, and/or NK cells in the cell population before and after contactwith the test compound. The phenotypes of the cells can be detected asdisclosed in the Examples and by similar techniques known to the skilledartisan. In some embodiments, the levels of ILCPs, ILC1, ILC2, ILC3,and/or NK cells after contact with the test compound is compared to anuntreated ILCP control.

In one embodiment, the invention encompasses a method for screening forcompounds that affect the development of ILCs comprising providing apopulation of innate lymphoid cell precursors (ILCPs), contacting thecell population with a test compound, and detecting a change in thephenotypes of the cells in the cell population.

In some embodiments, the test compound causes a reduction in thedifferentiation of the ILCPs. In some embodiments, the test compoundcauses an increase in the differentiation of the ILCPs. In someembodiments, the test compound causes a reduction in the differentiationinto a specific ILC subset. In some embodiments, the test compoundcauses an increase in the differentiation into a specific ILC subset.

In some embodiments, the method comprises combining the ILCPs with astimulus capable of differentiating them (e.g., OP9-DL4 culture system)and contacting the cell population with the test compound to determinethe effect of the compound on differentiation. In other embodiments, theeffect of the compound is determined in the absence of such a stimulusand/or with the addition of other compounds or stimuli (e.g. cytokines).

In some embodiments, the method comprises infusing a mouse with thepopulation of innate lymphoid cell precursors (ILCPs) and administeringthe test compound to the mouse. Preferably, the mouse is a humanizedmouse.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population have the phenotypeCD34-CD7+CD127+CD117+CD45RA+, lack the expression of NKp44 and RORyt,and are IL-1R1+ and CD69-, and/or optionally CD26+, and/or CD62L+.

Preferably, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the population have the phenotypeLin-CD34-CD7+CD127+CD117+CD45RA+, lack the expression of NKp44 andRORyt, and are IL-1R1+ and CD69-, and/or optionally CD26+, and/orCD62L+.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD7+CD127+CD117+CRTh2-.

In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%,95%, or 99% of the cells in the population areLin-CD94-CRTh2-CD127+CD117+.Most preferably, at least 25%, 35%, 50%,60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells in the population areCD127+CD117+CD3-CRTh2- . In some embodiments, at least 25%, 35%, 50%,60%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells in said population arefurther CD7+, NKp44-, CD94-, and/or Lin-, and/or further CD26+, and/orCD62L+. In some embodiments, at least 25%, 35%, 50%, 60%, 75%, 80%, 85%,90%, 95%, or 99% of the cells in the population areCD127+CD117+Lin-CRTh2- (wherein Lin- comprises CD3-), and optionallyfurther CD7+, NKp44-, and/or CD94-, and/or optionally further CD26+and/or CD62L+.Preferably, at least 75%, 80%, 85%, 90%, 95%, or 99% ofthe cells in the population are CD127+CD117+Lin-CRTh2- (wherein Lin-comprises CD3-), and optionally further CD7+, NKp44-, and/or CD94-,and/or optionally further CD26+ and/or CD62L+. In some preferredembodiments, at least 90 % of the cells in the population have thephenotype CD127+CD117+Lin-CRTh2-CD7+ or CD127+CD117+Lin-CRTh2-CD94-, andoptionally CD26+ and/or CD62L+; more preferably at least 99 % or 100 %of the cells in the population have the phenotypeCD127+CD117+Lin-CRTh2-CD94-, and optionally CD26+ and/or CD62L+.

EXAMPLES Material and Methods Human Blood and Tissues Samples

Blood samples from healthy donors were obtained from EstablissementFrançais du Sang (EFS, Paris) in an agreement signed with InstitutPasteur. Blood samples from patients with RORC mutation (RORC⁻/⁻-P1;RORC⁻/⁻-P2, p.A421X/Q421X) have been previously reported (26160376).Umbilical cord blood was collected from normal deliveries. Tonsils wereobtained from pediatric patients given tonsillectomy. Fetal liver wasobtained from elective abortion with gestational age ranging from 14 to20 weeks. Experiment with human fetal liver were approved by Medical andEthical Committees at Institut Pasteur and performed in full compliancewith French Law. Lungs were obtained from patients undergoing surgeryand samples were provided by Dr. JM Sallenave (Hôpital Bichat).Intestinal were obtained from colon cancer patients who underwentsurgery and provided by Dr. M Allez (Hôpital Saint Louis). Informedconsent was obtained from each patients as requested and approved by theinstitutional review boards of Necker Medical School, Paris DescartesUniversity, Hopital Bichat, Hopital Saint Louis, Assistance Publique -Hopital de Paris.

Human Immune System (HIS) Mice Model

BALB/c Rag2^(-/-)Il2rg^(-/-)Sirpa^(NOD) (BRGS) mice have been describedand were maintained in isolators at Institut Pasteur. CD34⁺HSC or CD117⁺ILC were sorted from peripheral blood of healthy donors using a FACSAria. Fetal liver CD34⁺HSC were isolated using CD34 Microbead Kit(Miltenyi). For in vivo transfer experiment, 1-3 x 10⁵ CD117⁺ ILC orCD34⁺ HSC were intrahepatically injected into sublethal irradiated (3Gy) new born (3-7 days-old) BRGS mice together with 0.3 µg of IL-2 and-7 (Miltenyi). Mice were received IL-2, -7, -1β, -23, -25 and -33 (0.3µg each) by intraperitoneal injection weekly and analyzed four weekspost-transplantation. For generation of HIS mice, fetal liver derivedCD34⁺ HSC were intrahepatically injected into sublethal irradiated (3Gy) new born (3-7 days-old) BRGS mice. Mice were sacrificed 8-9 weekspost-injection. Experiments were approved by ethical committee atInstitut Pasteur and validated by French Ministry of Education andResearch.

Cell Isolation From Blood, Tonsil, Gut, Fetal Liver and Lung

Human peripheral blood mononuclear cells (PBMC) from CB and PB wereisolated by Ficoll-Paque (GE Healthcare) density gradientcentrifugation. Single cell suspension from fetal liver and tonsil wasachieved by mechanical disruption through 70-µm filters. Lung andintestine samples were minced and digested with Liberase TL (25 µg/ml;Roche) and DNase I (50 µg/ml; Sigma-Aldrich) for 45 min in 37° C.shaking incubator. Digested tissues were passed through 70-µm filters.Lymphocytes from liver, lung and gut were isolated by Ficoll-Paquedensity gradient centrifugation.

FACS Analysis and Cell Sorting

For FACS analysis, cells were first stained with Flexible Viability DyeeFluor 506 (eBioscience) for 10 min followed by 20 min surfaceantibodies staining with Brilliant Stained Buffer (BD) on ice. Forexperiment involving intracellular TF staining, cells were fixed,permeabilized and stained using Foxp3/Transcription Factor StainingBuffer Kit (eBioscience). For intracellular cytokines staining, cellswere stimulated with PMA (10 ng/ml; Sigma) plus Ionomycin (1 µg/ml;Sigma) in the presence of Golgi Plug (BD) for 3 h. Cells were fixed,permeabilized and stained by Cytofix/Cytoperm Kit (BD). Samples wereacquired on LSRFortessa (BD) and analyzed by FlowJ10 (Tree Star).

For cell sorting from healthy PB, PBMC were first depleted of T cell, Bcell, pDC and monocytes by labeling with biotin-conjugated anti-CD3,anti-CD4, anti-CD19, anti-CD14, anti-CD123 followed by anti-biotinmicrobeads (Miltenyi) according to manufacturer’s instructions. Sortingfrom CB and tissues were performed with lineage depletion. Bulkpopulations were sorted to a purity ≥ 99% or as single cell indexsorting (both using FACSAria II; BD).

Bulk RNA Isolation, Library Construction, Sequencing and Analysis

10³ cells from each population were FACS sorted directly into 50 µl oflysis/binding buffer (Life Technologies). mRNA was captured withDynabeads oligo(dT)(Life Technologies), washed and eluted at 70° C. with10 µl of 10 mM Tris-Cl (pH7.5). A derivation of MARS-seq as described(24531970), developed for single-cell RNA-seq was used to produceexpression libraries with a minimum of two replicates per population. Anaverage of 4 million reads per library were sequenced and aligned tohuman reference genome (NCBI) using TopHat v2.0.10 with defaultparameter (19289445). Expression levels were calculated and normalizedfor each samples to the total number of reads using HOMER software(homer.salk.edu). It was focused on highly expressed genes with 2-folddifferential over the noise (8 reads) between the means of any twosubtypes. KEGG analysis was done by using DAVID (12734009).

Chromatin Immunoprecipitation and Sequencing (Chip-Seq) UsingChipMentation

FACS sorted cells (20-50 K) were immediately crosslinked in PBScontaining 1% formaldehyde (Sigma) for 10 min at room temperature forChIP—Seq analysis. Crosslinking was quenched by adding glycine (0.125 Mfinal concentration) followed by 5 min incubation at room temperature.Cells were placed on ice, washed with PBS and snap-frozen for storage at-80° C. Pellets were processed in parallel to minimize technicalvariation. Cells were resuspended in 100µl sonication buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH8 and 1x EDTA-free complete proteaseinhibitors; Roche) and transferred to a 0.65ml Bioruptor sonication tube(Diagenode). After 15 min incubation on ice, cells were sonicated for 30cycles (30 sec ON — 30 sec OFF) using a Bioruptor Pico sonicator(Diagenode) to shear chromatin down to ±250 bp fragments. Chromatin wasequilibrated by adding 900 µl 10x ChIP dilution buffer (0.01% SDS, 1.1%Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8, 167 mM NaCl) andincubated overnight at 4° C. with 1 µl of H3K4Me2-specific antibody(ab32356, Abcam) or normal rabbit IgG as a negative control (sc-2027,Santa Cruz). In addition, 20 µl of protein A Dynabeads (Thermo FisherScientific) per IP were blocked in PBS containing 0.1% BSA (Sigma) byincubation overnight at 4° C. The next day, beads were resuspended inthe original volume with ChIP dilution buffer and added to the chromatinextracts. After 2 hours of incubation at 4° C., beads were collected andwashed with Low Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mMTris-HCl pH8, 150 mM NaCl), High Salt buffer (0.1% SDS, 1% Triton X-100,2 mM EDTA, 20 mM Tris-HCl pH8, 500 mM NaCl) and LiCl buffer (10 mMTris-HCl pH8, 10 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% deoxycholicacid). Chromatin-antibody immobilized on magnetic beads were thensubjected to tagmentation as recently described (Schmidl et al., 2015).Eluted DNA was purified using MinElute spin columns (Qiagen) andamplified for 8-12 cycles using Nextera PCR primers. Libraries werepurified using dual (0.5x-2.0x) SPRI Ampure XP beads (Beckman Coulter),pooled (up to 10 per sequencing run) and sequenced on a NextSeq500(Illumina) running a single-read 75 bp protocol.

ChIP-Seq Data Processing, Analysis and Visualization

Reads were demultiplexed using BaseSpace (Illumina) and aligned to themouse genome (mm10 build) using Bowtie (Langmead, B. & Salzberg, S. L.Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359,doi:10.1038/nmeth.1923 (2012)) with standard settings, removing readsthat could not be uniquely mapped. Indexed and sorted bam files wereparsed to HOMER (Heinz, S. et al. Simple combinations oflineage-determining transcription factors prime cis-regulatory elementsrequired for macrophage and B cell identities. Mol Cell 38, 576-589,doi:10.1016j.molcel.2010.05.004 (2010)) for further analysis. Tagdirectories were generated for each sample with removal of duplicatereads (-tbp 1 option). BedGraph files displaying normalized counts(reads per million) were generated for direct visualization in the UCSCGenome Browser (https://genome.ucsc.edu/) using the makeUCSCfile HOMERscript. H3K4Me2 enriched regions were identified using HOMER findPeakswith -region -size 1000 -minDist 2500 options. Overlapping andnon-overlapping regions between two samples were identified using theintersect function of BEDTools (Quinlan, A. R. & Hall, I. M. BEDTools: aflexible suite of utilities for comparing genomic features.Bioinformatics 26, 841-842, doi:10.1093/bioinformatics/btq033 (2010)) orthe HOMER mergePeaks script (-d given option) requiring a minimaloverlap of 1bp. Sets of cell type-specific H3K4Me2+ regions werevisualized as heatmaps with Java TreeView (Saldanha, A. J. JavaTreeview--extensible visualization of microarray data. Bioinformatics20, 3246-3248, doi:10.1093/bioinformatics/bth349 (2004)). Regions/peakswere assigned to putative target genes GREAT (McLean, C. Y. et al. GREATimproves functional interpretation of cis-regulatory regions. NatBiotechnol 28, 495-501, doi:10.1038/nbt.1630 (2010)). GREAT wassubsequently used to calculate enrichments of these genes for knownpathway signatures using the whole genome as background.

Bulk and Single Cell Culture

All in vitro culture experiments were performed in Yssel’s medium(18432890) supplemented with 2% human AB serum (EFS). 2-3 x 10³ stromalcells were pre-seeded in 96-well round bottom plates one night beforeculture. Yssel’s medium is prepared in house by using IMDM (Invitrogen)plus 0.25 % (w/v) bovine serum albumin (Sigma), 1.8 µg/L 2-aminoethanol, 40 µg/L Apo-transferrin, 5 µg/L insulin andpenicillin/Streptomycin. For bulk culture, 100-300 FACS sorted cell wereplated on the stromal cells. For cloning experiment, cells wereindex-sorted directly into the 96-well plates pre-seeded with stromalcells. Cytokines IL-2, -7 (20 ng/ml each, Miltenyi), IL-12, -18, -25,-33, -1β, -23 (20 ng/ml each, R&D) were provided in various combinationsas indicated. For bulk culture, fresh cytokines and medium werereplenish every 5 days and analyzed after 10 days expansion. For cloningexperiment, cytokines and medium were replenished every 7 days andanalyzed after 14-18 days of culture.

Example 1: Characterization of Human Peripheral Blood CD117⁺ ILC

Data are represented as Median unless specified. The sample size foreach experiment and the replicate number of experiments are included inthe figure legends. Circulating ILC can be identified as a low frequencypopulation (< 0.2% of total CD45⁺ cells) within lineage-CD7⁺CD56⁻CD127⁺peripheral blood (PB) cells of healthy individuals as well as patientssuffering from diverse clinical syndromes ((Hazenberg and Spits, 2014;Munneke et al., 2014; FIGS. 1A and 1B). Further fractionation of PB ILCsinto ILC1, ILC2 and ILC3 has been achieved using phenotypic markers thatidentify ILC subsets in fetal tissues and tonsils, including CD161,CRTh2, CD117 and NKp44 (Spits et al., 2013). As such, previous reportshave identified circulating ILC2 (CD161⁺CRTh2⁺GATA-3⁺ cells) as well asILC1 (CD161⁺CRTh2⁻⁻CD117⁻⁻T-BET⁺ cells) in human blood (Mjösberg et al.,2011). Circulating ILCs also include a predominant CD117⁺ subset thatlacks CRTh2 expression ((Munneke et al., 2014; Vély et al., 2016); FIGS.1A and 1B). Previous studies have considered these cells as circulatingILC3since tissue-resident ILC3 strongly express CD117 (Cella et al.,2009; Cupedo et al., 2009). However, it was found that PB CD 117⁺ ILCdiffer dramatically from gut CD 117⁺ ILC in that they lack expression ofNKp44 and of the transcription factor (TF) RORyt that identifies ILC3(FIG. 1C). Accordingly, PB CD 117⁺ ILC do not produce IL-17A or IL-22after stimulation, whereas gut CD117⁺ cells abundantly produce theseILC3-associated cytokines (FIG. 1D). Interestingly, circulating CD117⁺ILC express high levels of IL-1R1, CD45RA and are CD69⁻, whereasgut-resident ILC3 are CD69⁺ but IL-1R1⁻ and CD45RA⁻ (FIG. 1C). Theseobservations suggest that PB CD 117⁺ ILC are not bona fide ILC3.

PB CD117⁺ ILC did not express signature TFs that characterize otherknown ILC subsets (ie.: T-BET, EOMES, GATA-3^(hi)) (FIG. 1C).Accordingly, PB CD117⁺ ILC failed to express markers associated with NKcells, ILC1 and ILC2, such as CD94, CD244, CRTh2 (FIG. 1A) and did notproduce IFN-γ or IL-13 after stimulation with pharmacological activators(FIG. 1D). Taken together, these results suggest that PB CD117⁺ ILC donot represent any canonical ILC subset.

Example 2: Transcription and Chromatin Landscapes of CD117⁺ ILC revealan ILC Precursor Profile

As CD117 is highly expressed on hemato-lymphoid progenitors (Ikuta andWeissman, 1992; Kikushige et al., 2008), it was hypothesized that PBCD117⁺ ILC might include uncommitted lymphoid precursors. In order tofurther understand the identity of PB CD 117⁺ ILC, the transcriptomicand epigenetic landscapes of highly purified circulating CD117⁺ ILC wasprofiled and compared to CD34⁺ HSC (FIG. 2A); the latter representingimmature hematopoietic progenitors with multi-lineage potential (Baum etal., 1992; Mohtashami et al., 2010).

Chromatin immunoprecipitation was performed followed by high-throughputsequencing using transposase-mediated tagmentation (ChIPm-Seq) thatallowed the inventors to directly analyse the epigenome of small numberof purified cells. To expose common and unique epigenetic features ofCD34⁺ HSC and CD117⁺ ILC, histone H3 lysine 4 dimethylation (H3KMe2) wasmapped since it marks both active and poised gene regulatory elements(GRE) with superior precision than other histone modifications (Zhang etal. 2012 Cell, Koche et al. Cell Stem Cell 2011). Around 18,000 and35,000 GRE were identified in CD117⁺ ILC and CD34⁺ HSC respectively(FIG. 2B), the majority of which were located in introns and intergenicregions (Supp. FIG. X). A significant number of H3K4Me2⁺ GRE were sharedbetween the two cell types: 89% of GRE identified in CD117⁺ ILC showedsimilar enrichment in HSC and were associated with 13159 genes of whichmany encoded housekeeping functions. Nevertheless, 11% of H3K4Me2⁺ GREdetected in CD117⁺ ILC were absent in CD34⁺ HSC, potentially regulating2283 genes. Pathway analysis of these genes revealed a strong enrichmentfor immune system and lymphocyte related processes (FIG. 2C). Forexample, cytokine/chemokine signaling genes critical for lymphoiddevelopment and function such as IL1R1, IL7R, IL2RA/B were linked to aCD117⁺ ILC-specific GRE. Conversely, GRE only active in CD34⁺ HSC (54%of all GRE in CD34⁺ HSC) were located near genes involved in moregeneral pathway important for hematopoiesis, including hemostasis,platelet activation and Notch signaling pathway (FIG. 2C).

To compare the transcriptome of CD117⁺ ILC and CD34⁺ HSC, RNA sequencing(RNA-Seq) was performed. Clear differences in gene expression profilesemerged, with a large cluster of 1540 genes expressed at substantiallyhigher levels in CD117⁺ ILC (FIG. 2D). Among these were many genesstrongly linked to the lymphoid lineage, including IKZF1, CD2, CD7 andIL7R (FIG. 2D). In contrast, CD34⁺ HSC cells highly expressed genesinvolved in the broad development of diverse hematopoietic lineages,including GATA1, GATA2 and MYB (FIG. 2D) as well as cytokine receptorsfor myeloid lineages (CSF3R, CSF2RB, FLT3). Compared to HSC, CD117⁺ ILCexpress high levels of TF that have been shown to be essential formurine ILC development, including ID2, GATA3, TOX and TCF7. Transcriptscharacteristic of T and B cells development, such as RAG1, RAG2, EBF1,CD3E, ECL11A or LMO2 were not detected in CD117⁺ ILC although some ofthese genes are expressed by HSC.

As both transcriptomic and epigenetic analyses of CD117⁺ ILC identifiedstrong lymphoid signatures, these datasets were intersected in order togain insights into the developmental status of CD117⁺ ILC. A substantialproportion (26%) of the genes most highly expressed in CD117⁺ ILC werelocated in the direct vicinity of a CD117⁺ ILC-specific GRE (FIG. 2E).Surprisingly, these included many transcription factors previouslyimplicated in mouse ILC development, including ID2, GATA3, ETS1, TOX,TCF7, RORA and NOTCH1 (FIG. 2E) — consistent with the commitment ofCD117⁺ ILC to the innate lymphoid fate. In contrast, notable expressionlevels were not detected for any of the mature ILC TFs (EOMES, TBX21,RORC), cytokine receptors (CCR6, IL1RL1, IL23R) or signature cytokines(IFNG, IL13, IL5, IL22, IL17A). However, several of these mature ILCidentity genes were already marked with H3K4Me2, demonstrating that theyreside in a poised state (FIG. 2E). In contrast, key regulators of B andT cell development (RAG1, RAG2, EBF1, BCL11A, HES1, LMO2) were notselectively marked with H3K4Me2. Together, these analyses suggest thatCD117⁺ ILC represent lymphoid-biased progenitors carrying a TFexpression profile resembling a multi-potent ILC precursor (ILCP) withkey mature ILC signature genes in an epigenetically poised state.

Example 3: Peripheral Blood CD117⁺ ILC include multi-potent ILCprecursors (ILCP)

In order to assess the hematopoietic potential of circulating CD117⁺ILC, these cells were bulk cultured in the presence of variouscytokines. As CD117⁺ ILC express CD25, CD127 and CD121a (IL-1R1) (FIGS.1A, 1C), IL-2, IL-7 and/or IL-1β were added to these cultures. Whilebulk cultures minimally expanded in the presence of IL-2 and IL-7,robust proliferation was observed when cells were cultured in IL-1β(FIG. 3A). The additional presence of cytokines that can drive ILC1/NK(IL-12, -18), ILC2 (IL-25, -33) or ILC3 (IL-23) development did notfurther increase cell yield over that obtained with IL-1β (FIG. 3A).Bulk cultured cells did not harbor B (CD19⁺) or T (CD3⁺CD5⁺) cells butcomprised a pure population of CD7⁺ cells that were CD161⁺ and expressedvariable levels of CD117 and CD25 (FIG. 3B).

Remarkably, expanded cells included some EOMES⁺CD94⁺ NK cells as well ascells representing the three canonical ILC groups: IFN-γ⁺ ILC1, IL-13⁺ILC2 and NKp44⁺IL-17A⁺IL22⁺ ILC3 (FIG. 3B). While IL-25 and IL-33supplementation did not appreciably alter the distribution of ILCsubsets in these cultures, the addition of IL-12 clearly promoted thedevelopment of EOMES⁺CD94⁺ IFN-γ-producing NK cells and IL-23 wascritical for IL-17A-producing-ILC3 (FIG. 3B). These results not onlydefine a cytokine ‘mix’ that supports multi-lineage ILC and NK cellgeneration (IL-2, IL-7, IL-1β, IL-23) but also suggest that PB CD117⁺ILC harbors multi-lineage ILC precursors (ILCP).

The multi-lineage potential of circulating CD117⁺ ILC was furthercharacterized using a modified stromal cell-based culture system that ispermissive for B cell, T cell and myeloid cell development (FIG. 3C;Mohtashami et al., 2010)). Moreover, this system can extensively expandhuman NK cells and ILC subsets at the clonal level with minimalplasticity (Lim et al., 2016). Progeny of single PB CD117⁺ ILC culturedon OP9 and OP9-DL4 were analyzed to identify EOMES⁺ NK cells and ILCsubsets producing IFN-γ, IL-13, IL-17A and/or IL-22 (FIG. 3D). OP9-DL4stroma express a strong Notch ligand allowing the inventors to assessthe impact of triggering this pathway (Mohtashami et al., 2010). Theinventors’ analysis of over 340 clonal cultures allows several points tobe made. First, PB CD117⁺ ILC represent a heterogeneous population ofuni-potent and multi-potent ILC precursors (ILCP). Roughly half of thecultures derived from single CD117⁺ ILC generate a single ILC subset(ILC1, ILC2 or ILC3 only) and therefore represent lineage-restrictedILCP, whereas the remainder are multi-potent ILCP that can give rise to2 or more separate Lin⁻CD7⁺ ILC lineages (FIG. 3E). B cell and T cellpotential was not observed. Second, within the multi-potent ILCPpopulation, a substantial fraction (between 9-17%) are able to generateall three ILC subsets and likely represent immature uncommitted ILCP.Moreover, clonal IFN-γ⁺ cultures also comprise EOMES⁺ NK cellsdemonstrating that some PB ILCP have the potential to generate both‘helper’ and ‘cytotoxic’ ILC lineages at the single cell level. Third, asubset of Lin⁻CD7⁺ ILC clones failed to produce any cytokine tested(FIG. 3E). As these clones maintained high level of CD7 and CD117 butlacked other ILC markers, they may represent ILCP that have not furtherdifferentiated. Fourth, Notch signals clearly influence the cell fatepotential of CD117⁺ ILCP as multi-potentiality and development ofILC3-containing cells was enhanced on OP9-DL4 (FIG. 3E). Together, thesedata identify PB CD117⁺ ILC as a circulating pool of committed ILCprogenitors. The comparison of bulk and clonal assays clearlydemonstrate the importance of the single cell approach to defineheterogeneity of CD117⁺ ILC cell fate potential and to establishfunctional multipotency.

Example 4: Circulating CD117⁺ ILCP have multi-ILC Potential in Vivo inHumanized Mice

The in vivo potential of PB CD117⁺ ILCP was next assessed. Previousstudies have demonstrated the capacity of severely immunodeficient mousestrains engrafted with human CD34⁺ hematopoietic stem cell (HSC)progenitors to generate human lymphoid (B, T, NK) and myeloid (DC,macrophage, neutrophils) cell subsets (reviewed in (Shultz et al.,2012)). BALB/c Rag2^(-/-)Il2rg^(-/-)Sirpa^(NOD) (BRGS) mice that arepermissive for robust multi-lineage human hematopoietic cell engraftmentwere used (Legrand et al., 2011). Human PB CD34⁺ HSC and CD117⁺ ILCPfrom same donors were adoptively transferred to newborn BRGS mice;cytokine supplementation (IL-2, IL-7, IL-1β, IL-23) was provided andmice were analyzed 4 weeks later (FIG. 4A). BRGS mice engrafted withhuman CD34⁺ HSC developed CD19⁺ B cells and CD14/CD33⁺ myeloid cells inthe bone marrow, while CD3/CD5⁺ T cells and Lin⁻CD7⁺ NK/ILC weredetected in the gut (FIG. 4B). In contrast, BRGS mice receiving PBCD117⁺ ILCP developed Lin⁻CD7⁺ cells but no myeloid cells, B cells or Tcells. Human CD45⁺ progeny from transferred CD117⁺ ILCP were detected inmultiple organs, including the spleen, lung, gut and liver (FIG. 4C). Ateach of these tissue sites, EOMES⁺ NK cells as well as diverse CD127⁺ILC subsets could be identified that produced IFN-γ, IL-13, IL-17Aand/or IL-22 ex vivo upon stimulation (FIG. 4C). These resultsdemonstrate that PB CD117⁺ ILCP have the potential to generate all knownILC subsets and NK cells in vivo. Since PB CD117⁺ ILCP lack myeloid, Band T cell potential, it was concluded that these cells comprisecommitted ILC progenitors.

Example 5: Human CD117⁺ ILCP develop from CD34⁺ HSC in Vivo

The developmental relationship between CD34⁺ HSC and CD117⁺ ILCP wasnext interrogated. Immunodeficient neonatal BRGS mice were engraftedwith purified CD34⁺ HSC and were sacrificed 8-9 weeks later (FIG. 5A).Human CD45⁺ cells were analyzed in bone marrow, lung, liver and spleen.As expected (Legrand et al., 2011), these different tissue sitesharbored human CD45⁺ cells, including a variety of lineage⁺ T, B andmyeloid cells (FIG. 5B, data not shown). Moreover, within the subset ofLin⁻CD7⁺ cells, a clearly defined subpopulation of CD127⁺CD117⁺ cellscould be discerned in multiple tissues that lacked T-BET and EOMESexpression (FIG. 5B). These included CD127⁺CD117⁺ cells that expressedlow levels of GATA-3 and RORyt and were NKp44⁻ (FIG. 5D) and thereforeresembled PB CD117⁺ ILCP. Ex vivo stimulation failed to elicit cytokineproduction from CD127⁺CD117⁺ cells (FIG. 5E). These cells were sortedand bulk cultured in the presence of IL-2, IL-7, IL-1β and IL-23.Expanded cells contained subsets able to produce IFN-γ, IL-13, IL-17Aand IL-22 (FIG. 5E) thereby confirming the presence of human ILCP. Theseresults demonstrate that CD34⁺ HSC can give rise to CD117⁺ ILCP in vivo.

Example 6: Human CD117⁺ ILCP are Present in Fetal Liver, Cord Blood andAdult Lung

The stage of development when human CD117⁺ ILCP arise was next assessed.Human ILC subsets in fetal liver (FL) were first studied as this organhas been shown to harbor several immature hematopoietic precursorpopulations (Rollini et al., 2007) and is proposed as a sight for thedevelopment of lymphoid tissue inducer cells in the mouse (Cherrier etal., 2012). Lin⁻CD127⁺ ILC within FL contain a predominant CD117⁺subset. Interestingly, these cells express RORyt at levels exceedingtheir peripheral blood counterparts (FIG. 1C) and moreover express CCR6,Neuropilin-1 (NRP-1) but not NKp44. Despite these differences, FL CD117⁺ILC did not produce significant amounts of IL-17A or IL-22 afterstimulation suggesting that they were not fully mature ILC3.Nevertheless, when FL CD117⁺ ILC were expanded in vitro,IL-17A-producing ILC3 were abundantly generated. Moreover, IL17A⁺ ILC3developed on stromal cells lacking DL4 suggesting that additional Notchengagement was not necessary for this process (FIG. 6A). Interestingly,bulk cultures of FL CD117⁺ ILC also contain detectable IL-13- andIFN-γ-producing cells, although at lower frequency. Clonal analysisrevealed that FL CD117⁺ ILC harbor, as expected, a high proportion ofILC3 committed progenitors. Still, a substantial fraction of thispopulation includes multi-potent ILCP (FIGS. 6B, C) that are moreclearly revealed in the presence of Notch ligands. These resultsdemonstrate that the human FL harbors CD34⁻ CD127⁺CD117⁺ multi-potentILCP that can generate all known ILC subsets. The enrichment ofILC3-committed progenitors in this tissue site suggests thatenvironmental signals may direct the further specification ofmulti-potent ILCP towards an ILC3 fate during this period.

CD117⁺ ILC from human cord blood (CB) were next characterized. Liketheir PB counterparts, CB CD117⁺ ILC lacked NKp44 expression as well asthat of CCR6 and NRP-1 and were CD45RA⁺. Moreover, CB CD117⁺ ILC failedto express RORyt and T-BET but were GATA-3^(lo), thus resembling PBILCP. Like PB CD117⁺ ILC, CB CD117⁺ ILC did not produce cytokines(IFN-γ, IL-13, IL-17A or IL-22) ex vivo after stimulation. However, bulkculture of CB CD117⁺ ILC in IL-2, IL-7, EL-1β and IL-23 generateddiverse cytokine-producing ILC subsets that included IFN-γ⁺ ILC1, IL-13⁺ILC2 and IL-17A⁺ or IL-22⁺ ILC3 (FIG. 6D). No T, B or myeloid cells weredetected in cultures of CB CD117⁺ ILC (data not shown). Further clonalanalysis revealed that CB CD117⁺ ILC harbored a diverse mix ofuni-potent and multi-potent ILCP (FIGS. 6E and 6F). Unlike FL CD117⁺ILC, CB CD117⁺ ILC were not biased towards ILC3-committed progenitors,but more closely resembled PB CD117⁺ ILCP. As for ILCP from PB or FL,Notch stimulation resulted in an enhanced frequency of multi-potent ILCP(especially those having the potential for IL-17A⁺ and IL-22⁺ ILC3) andreduced the frequency of cytokine⁻ ILC clones, suggesting that thispathway facilitated directed development of specific ILC subsets.

The phenotype and potential of CD117⁺ ILC from adult lung tissue wasalso examined. Lung CD117⁺ ILC harbored discreet populations of NKp44⁺and RORγt⁺ ILC but were largely CD45A⁻. Bulk cultures of lung CD117⁺ ILCgenerated diverse cytokine-producing ILC subsets and EOMES⁺ NK cells(FIG. 6G); further analysis using clonal assays defined the NKp44⁻fraction of lung CD117⁺ ILC as a mixture of uni-potent and multi-potentILCP (FIGS. 6H and 6I). These results demonstrate that a variety ofILCP, including multi-potent progenitors, are present in human mucosaltissues.

Example 7: ILC Precursors Reside Within Secondary Lymphoid Tissues

Human secondary lymphoid tissues (lymph nodes, tonsils) harbor diverseILC subsets and their precursors (Bernink et al., 2013; Cella et al.,2009; Fehniger et al., 2003; Mjösberg et al., 2011; Renoux et al., 2015;Montaldo et al., 2015; Scoville et al., 2016). It was therefore ofinterest to further characterize tonsillar CD117⁺ ILCP and to assesstheir cell fate potential. CD117⁺ ILC from pediatric tonsils harbor apredominant NKp44⁺ ILC3 subset that can be stimulated to produce IL-17Aand IL-22 (Hoorweg et al., 2012). This population also appears to haveextensive functional plasticity as stimulation (using IL-1β, IL-12,IL-23) can modify cytokine outputs of these cells (Bernink et al., 2015;Bernink et al., 2013; Cella et al., 2010). Within tonsillar CD117⁺ ILC,it was found that NKp44⁻ cells were CD45RA⁺ and NRP-1⁻, while NKp44⁺cells were CD45RA⁻ and NRP-1⁺. These suggest that NKp44⁺ ILC3 are moremature and differentiate from NKp44⁻ cells (Bernink et al., 2015).However, cytokine production profiles were different in bulk culturesfrom tonsillar NKp44⁻ versus NKp44⁺ CD117⁺ ILC (FIGS. 6J and 6M). Inparticular, IFN-γ⁺ cells and IL-13⁺ cells were more obvious in culturesderived from NKp44⁻ cells, especially on OP9 stroma (FIG. 6J).

In order to better understand the relationship between NKp44⁻ and NKp44⁺CD117⁺ ILC, clones from both subsets were generated and theircytokine-production potential analyzed. Striking differences wereobserved. Clones derived from NKp44⁺ CD117⁺ ILC were highly enrichedILC3 producing IL-17A and/or IL-22 (FIGS. 6N and 6O). A fraction ofclones co-expressed IFN-γ (14%) that likely represent ‘plastic’ ILC3that may up-regulate T-BET as previously shown (Bernink et al., 2015).In contrast, clones derived from NKp44⁻ CD117⁺ ILC were quiteheterogeneous with cells producing not only IL-22 and/or IL-17A but alsoabundant single IFN-γ⁺ clones as well as single IL-13⁺ clones (FIGS. 6Kand 6L) that were not detected from NKp44⁺ CD117⁺ ILC (FIGS. 6N and 6O).The fact that IFN-γ⁺ ILC1 clones were observed was unexpected given theprevious reports that tonsillar CD127⁺ ILC1 differentiate into IL-22producing ILC3 in the presence of IL-2, IL-23 and IL-1β (Bernink et al.,2015). Lastly, multi-potent ILCP giving rise to three ILC subsets wereonly found in NKp44⁻ CD117⁺ ILC. Taken together, these results suggestthat tonsillar CD117⁺ ILC are quite heterogeneous comprising NKp44⁻ ILCPas well as NKp44⁺ ILC3. Furthermore, the inventors’ use of clonal assaysclearly allows the definition of ILCP repertoires that is not visualizedat the bulk culture level.

Example 8: RORC-deficient Patients Harbor ILCP but Fail to GenerateIL-17A⁺ ILC3

A committed ILCP in human secondary lymphoid tissue with aCD34⁺CD45RA⁺CD117⁺ phenotype was shown to highly express the TF RORC(Scoville et al., 2016). As CD117⁺ ILCP are developmentally downstreamfrom CD34⁺ HSC (FIG. 5 ), it is possible that the previously describedCD34⁺CD45RA⁺CD117⁺ ILCP subset is an intermediate in this pathway. Inorder to address whether RORC was required for generation of humanCD117⁺ ILCP RORC-deficient patients were studied. RORC deficiency inhumans is associated with mucocutaneous candidiasis and previous studiesdemonstrated that this TF is essential for differentiation of Th17 cellsthat protect against fungal pathogens (Okada et al., 2015). ILC subsetsfrom peripheral blood cells from 2 patients with RORC deficiency werestudied (FIG. 7A). Lin⁻CD7⁺ cells contained a predominant population ofCD56⁺ NK cells (both CD56^(bright) and CD56^(dim)) in both controls andRORC-deficient patients and a discreet subpopulation of CD127⁺ ILCs wasalso clearly detected. As previously described (Okada et al., 2015), areduction in the frequency of CD117-expressing ILC was noted in patientswith RORC deficiency, although this population was still clearly present(FIGS. 7A and 7B). In contrast, ILC1 were present and the percentage ofILC2 from total ILC was significantly increased in the absence of RORC(FIG. 7B). Sorted CD117⁺ ILC from control and RORC-deficient patientswere bulk cultured as described above. Robust growth of Lin⁻CD7⁺ cellswas observed with no significant difference between WT andRORC-deficient cells. Diverse cytokine producing cells were identifiedin these cultures, including those producing IFN-γ, IL-13 or IL-22,however, no IL-17A-producing cells were present (FIG. 7C). Developmentof EOMES⁺IFN-γ⁺ NK cells was not affected by the absence of RORC. Theseresults demonstrate that RORC is not required for the development of NKcells, ILC1, ILC2 or IL-22⁺ ILC3 but is essential for the generation ofIL-17A⁺ ILC3 from ILCP in humans.

Example 9: Analysis of ILCP Markers in Blood

While CD127 and CD117 are expressed by ILCP, they are not specific. Togain insights into minimal essential markers, combinations of markersthat could be used to highly enrich for ILCP were analysed. Therefore,percentages of different cell types was determined by FACS usingdifferent markers to isolate cells from adult peripheral blood. Theresults present analysis of the enrichment for human ILCP in peripheralblood using multi-parametric FACS analysis. Using the different gatingscheme, one can estimate the enrichment of ILCP as well as other celltypes (T cells, NK cells, ILC2) that may be present. The results arepresented in FIGS. 8A-F and FIGS. 9A-G.

Sorting using the markers CD127+CD117+ cells generated about 20% ILCPand 60-80% T cells (FIGS. 8A and 9A). Including CD7, excluding CRTh2 ora combination of both were not sufficient to enrich ILCP withinCD127+CD117+ cells since contaminating T cells predominated (FIGS. 8A-D:FIGS. 9A-D). Excluding Lineage cells (including CD3) strongly enrichedILCP in CD127+CD117+ cells (FIGS. 8E-F; FIGS. 9E-G). Exclusion of CRTh2ILC2 in Lin-CD 127+CD117+ cells provided further ILCP enrichment,generating at least 75 % of ILCP (FIG. 9E). CD7 was not required forthis effect (FIGS. 8E and 9F). Excluding CD94 allowed isolation of pureILCP from Lin-CD127+CD117+CRTh2- cells (FIGS. 8F and 9G). Accordingly,sorting using the markers CD127+CD117+CD7+CRTh2-Lin- generatedapproximately 90% ILCP (FIGS. 8E and 9F) and sorting using the markersCD127+CD117+CRTh2-Lin-CD94- generated approximately 100% ILCP (FIGS. 8Fand 9G).

Example 10: Additional ILCP Markers

Human ILCP (defined as Lin-CD127+CD117+ CRTh2-) were additionallyscreened for expression of additional cell surface markers that could beuseful surrogates for isolating these cells. Variable CD62L and CD26expression were identified on human ILCP with most cells being CD62L+and a large proportion of cells expressing CD26 (FIG. 10 ).

Clonal analysis demonstrated that all of these subsets harboredmulti-potent ILCP (Table 1).

TABLE 1 Clonal analysis of CD62L and CD26 human ILCP subsets. ILCPCD62L+CD26- ILCP CD62L+CD26+ ILCP CD62L-CD26- ILCP CD62L-CD26+ Nocytokine 21; 33 % 2; 3 % 13; 42 % 10; 16 % Multipotent 7; 11 % 25; 40 %3; 10 % 20; 32 % ILC1 2; 3 % 3; 5 % 2; 6 % 3; 5 % ILC2 29; 46 % 25; 41 %11; 36 % 28; 44 % ILC3 0; 0 % 0; 0 % 0; 0 % 0; 0 % NK 4; 7 % 7; 11% 2; 6% 2; 3% Indicated human ILCP subsets were sorted as single cells andcultured on OP9 stroma supplemented with human IL-1b, IL-2, IL-7 andIL-23. Clones were then analyzed for cytokine production (IFNg, IL-13,IL-17A, IL-22) after 3 hr stimulation with PMA/ionomycin. Frequencies ofuni-potent and multipotent ILCP are indicated. Putative ILCP areidentified as ‘No cytokine’.

These results identify additional ‘optional’ markers (CD62L, CD26) thatcan be used to isolate subsets of human ILCP.

Human ILCP expansion: Analysis of human ILCP clones (using OP9 stromalcells and combinations of IL-2, IL-7, IL-1β and IL-23 identified cellsthat failed to express any tested cytokines (IFN-g, IL-13, IL-17A,IL-22). These ILCP ‘clones’ had expanded between 100- and 1000-fold innumber (FIG. 11 ). These cells were phenotypes and found to expressCD117, CD45RA and CD26 but were CD62L- (FIG. 12 ). Recloning experimentsshowed that these cells continued to have multi-potency for all ILCsubsets. It was concluded that human ILCP can be expanded usingcombinations of stromal cells and cytokines and retain functionalproperties.

REFERENCES

Artis, D., and Spits, H. (2015). The biology of innate lymphoid cells.Nature 517, 293-301.

Baum, C.M., Weissman, I.L., Tsukamoto, A.S., Buckle, A.M., and Peault,B. (1992). Isolation of a candidate human hematopoietic stem-cellpopulation. Proc Natl Acad Sci U S A 89, 2804-2808.

Bernink, J.H., Krabbendam, L., Germar, K., de Jong, E., Gronke, K.,Kofoed-Nielsen, M., Munneke, J.M., Hazenberg, M.D., Villaudy, J.,Buskens, C.J., et al. (2015). Interleukin-12 and -23 Control Plasticityof CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the IntestinalLamina Propria. Immunity 43, 146-160.

Bernink, J.H., Peters, C.P., Munneke, M., te Velde, A.A., Meijer, S.L.,Weijer, K., Hreggvidsdottir, H.S., Heinsbroek, S.E., Legrand, N.,Buskens, C.J., et al. (2013). Human type 1 innate lymphoid cellsaccumulate in inflamed mucosal tissues. Nat Immunol 14, 221-229.

Cella, M., Fuchs, A., Vermi, W., Facchetti, F., Otero, K., Lennerz,J.K., Doherty, J.M., Mills, J.C., and Colonna, M. (2009). A humannatural killer cell subset provides an innate source of IL-22 formucosal immunity. Nature 457, 722-725.

Cella, M., Otero, K., and Colonna, M. (2010). Expansion of human NK-22cells with IL-7, IL-2, and IL-1beta reveals intrinsic functionalplasticity. Proc Natl Acad Sci U S A 107, 10961-10966.

Chea, S., Perchet, T., Petit, M., Verrier, T., Guy-Grand, D., Banchi,E.G., Vosshenrich, C.A., Di Santo, J.P., Cumano, A., and Golub, R.(2016a). Notch signaling in group 3 innate lymphoid cells modulatestheir plasticity. Sci Signal 9, ra45.

Chea, S., Schmutz, S., Berthault, C., Perchet, T., Petit, M.,Burlen-Defranoux, O., Goldrath, A.W., Rodewald, H.R., Cumano, A., andGolub, R. (2016b). Single-Cell Gene Expression Analyses RevealHeterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors toNotch Signaling. Cell Rep 14, 1500-1516.

Cherrier, M., Sawa, S., and Eberl, G. (2012). Notch, Id2, and RORγtsequentially orchestrate the fetal development of lymphoid tissueinducer cells. J Exp Med 209, 729-740.

Constantinides, M.G., McDonald, B.D., Verhoef, P.A., and Bendelac, A.(2014). A committed precursor to innate lymphoid cells. Nature 508,397-401.

Cupedo, T., Crellin, N.K., Papazian, N., Rombouts, E.J., Weijer, K.,Grogan, J.L., Fibbe, W.E., Cornelissen, J.J., and Spits, H. (2009).Human fetal lymphoid tissue-inducer cells are interleukin 17-producingprecursors to RORC+ CD127+ natural killer-like cells. Nat Immunol 10,66-74.

Dallas, M.H., Varnum-Finney, B., Delaney, C., Kato, K., and Bernstein,I.D. (2005). Density of the Notch ligand Delta1 determines generation ofB and T cell precursors from hematopoietic stem cells. J Exp Med 201,1361-1366.

Diefenbach, A., Colonna, M., and Koyasu, S. (2014). Development,differentiation, and diversity of innate lymphoid cells. Immunity 41,354-365.

Eberl, G., Colonna, M., Di Santo, J.P., and McKenzie, A.N. (2015).Innate lymphoid cells. Innate lymphoid cells: a new paradigm inimmunology. Science 348, aaa6566.

Fuchs, A., Vermi, W., Lee, J.S., Lonardi, S., Gilfillan, S., Newberry,R.D., Cella, M., and Colonna, M. (2013). Intraepithelial type 1 innatelymphoid cells are a unique subset of IL-12- and IL-15-responsiveIFN-γ-producing cells. Immunity 38, 769-781.

Gasteiger, G., Fan, X., Dikiy, S., Lee, S.Y., and Rudensky, A.Y. (2015).Tissue residency of innate lymphoid cells in lymphoid and nonlymphoidorgans. Science 350, 981-985.

Hazenberg, M.D., and Spits, H. (2014). Human innate lymphoid cells.Blood 124, 700-709.

Hoorweg, K., Peters, C.P., Cornelissen, F., Aparicio-Domingo, P.,Papazian, N., Kazemier, G., Mjösberg, J.M., Spits, H., and Cupedo, T.(2012). Functional Differences between Human NKp44(-) and NKp44(+)RORC(+) Innate Lymphoid Cells. Front Immunol 3, 72.

Ikuta, K., and Weissman, I.L. (1992). Evidence that hematopoietic stemcells express mouse c-kit but do not depend on steel factor for theirgeneration. Proc Natl Acad Sci U S A 89, 1502-1506.

Juelke, K., and Romagnani, C. (2016). Differentiation of human innatelymphoid cells (ILCs). Curr Opin Immunol 38, 75-85.

Kikushige, Y., Yoshimoto, G., Miyamoto, T., Iino, T., Mori, Y., Iwasaki,H., Niiro, H., Takenaka, K., Nagafuji, K., Harada, M., et al. (2008).Human Flt3 is expressed at the hematopoietic stem cell and thegranulocyte/macrophage progenitor stages to maintain cell survival. JImmunol 180, 7358-7367.

Kim, B.S., and Artis, D. (2015). Group 2 innate lymphoid cells in healthand disease. Cold Spring Harb Perspect Biol 7.

Klose, C.S., Flach, M., Möhle, L., Rogell, L., Hoyler, T., Ebert, K.,Fabiunke, C., Pfeifer, D., Sexl, V., Fonseca-Pereira, D., et al. (2014).Differentiation of type 1 ILCs from a common progenitor to allhelper-like innate lymphoid cell lineages. Cell 157, 340-356.

Koues, O.I., Collins, P.L., Cella, M., Robinette, M.L., Porter, S.I.,Pyfrom, S.C., Payton, J.E., Colonna, M., and Oltz, E.M. (2016). DistinctGene Regulatory Pathways for Human Innate versus Adaptive LymphoidCells. Cell 165, 1134-1146.

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment withBowtie 2. Nat Methods 9, 357-359.

Legrand, N., Huntington, N.D., Nagasawa, M., Bakker, A.Q., Schotte, R.,Strick-Marchand, H., de Geus, S.J., Pouw, S.M., Böhne, M., Voordouw, A.,et al. (2011). Functional CD47/signal regulatory protein alpha(SIRP(alpha)) interaction is required for optimal human T-and naturalkiller- (NK) cell homeostasis in vivo. Proc Natl Acad Sci U S A 108,13224-13229.

Lim, A.I., Menegatti, S., Bustamante, J., Le Bourhis, L., Allez, M.,Rogge, L., Casanova, J.L., Yssel, H., and Di Santo, J.P. (2016). IL-12drives functional plasticity of human group 2 innate lymphoid cells. JExp Med 213, 569-583.

Luci, C., Reynders, A., Ivanov, I.I., Cognet, C., Chiche, L., Chasson,L., Hardwigsen, J., Anguiano, E., Banchereau, J., Chaussabel, D., et al.(2009). Influence of the transcription factor RORgammat on thedevelopment of NKp46+ cell populations in gut and skin. Nat Immunol 10,75-82.

Mjösberg, J.M., Trifari, S., Crellin, N.K., Peters, C.P., van Drunen,C.M., Piet, B., Fokkens, W.J., Cupedo, T., and Spits, H. (2011). HumanIL-25- and IL-33-responsive type 2 innate lymphoid cells are defined byexpression of CRTH2 and CD161. Nat Immunol 12, 1055-1062.

Mohtashami, M., Shah, D.K., Nakase, H., Kianizad, K., Petrie, H.T., andZúñiga-Pflücker, J.C. (2010). Direct comparison of Dll1- andDll4-mediated Notch activation levels shows differential lymphomyeloidlineage commitment outcomes. J Immunol 185, 867-876.

Montaldo, E., Juelke, K., and Romagnani, C. (2015). Group 3 innatelymphoid cells (ILC3s): Origin, differentiation, and plasticity inhumans and mice. Eur J Immunol 45, 2171-2182.

Montaldo, E., Teixeira-Alves, L.G., Glatzer, T., Durek, P., Stervbo, U.,Hamann, W., Babic, M., Paclik, D., Stölzel, K., Gröne, J., et al.(2014). Human RORγt(+)CD34(+) cells are lineage-specified progenitors ofgroup 3 RORyt(+) innate lymphoid cells. Immunity 41, 988-1000.

Munneke, J.M., Björklund, A.T., Mjösberg, J.M., Garming-Legert, K.,Bernink, J.H., Blom, B., Huisman, C., van Oers, M.H., Spits, H.,Malmberg, K.J., et al. (2014). Activated innate lymphoid cells areassociated with a reduced susceptibility to graft-versus-host disease.Blood 124, 812-821.

Okada, S., Markle, J.G., Deenick, E.K., Mele, F., Averbuch, D., Lagos,M., Alzahrani, M., Al-Muhsen, S., Halwani, R., Ma, C.S., et al. (2015).IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacteriumin humans with bi-allelic RORC mutations. Science 349, 606-613.

Rollini, P., Faes-Van’t Hull, E., Kaiser, S., Kapp, U., and Leyvraz, S.(2007). Phenotypic and functional analysis of human fetal liverhematopoietic stem cells in culture. Stem Cells Dev 16, 281-296.

Sawa, S., Cherrier, M., Lochner, M., Satoh-Takayama, N., Fehling, H.J.,Langa, F., Di Santo, J.P., and Eberl, G. (2010). Lineage relationshipanalysis of RORgammat+ innate lymphoid cells. Science 330, 665-669.

Schmidl, C., Rendeiro, A.F., Sheffield, N.C., and Bock, C. (2015).ChIPmentation: fast, robust, low-input ChIP-seq for histones andtranscription factors. Nat Methods 12, 963-965.

Scoville, S.D., Mundy-Bosse, B.L., Zhang, M.H., Chen, L., Zhang, X.,Keller, K.A., Hughes, T., Cheng, S., Bergin, S.M., Mao, H.C., et al.(2016). A Progenitor Cell Expressing Transcription Factor RORytGenerates All Human Innate Lymphoid Cell Subsets. Immunity 44,1140-1150.

Seehus, C.R., Aliahmad, P., de la Torre, B., Iliev, I.D., Spurka, L.,Funari, V.A., and Kaye, J. (2015). The development of innate lymphoidcells requires TOX-dependent generation of a common innate lymphoid cellprogenitor. Nat Immunol 16, 599-608.

Serafini, N., Vosshenrich, C.A., and Di Santo, J.P. (2015).Transcriptional regulation of innate lymphoid cell fate. Nat Rev Immunol15, 415-428.

Shih, H.Y., Sciumè, G., Mikami, Y., Guo, L., Sun, H.W., Brooks, S.R.,Urban, J.F., Davis, F.P., Kanno, Y., and O’Shea, J.J. (2016).Developmental Acquisition of Regulomes Underlies Innate Lymphoid CellFunctionality. Cell 165, 1120-1133.

Shultz, L.D., Brehm, M.A., Garcia-Martinez, J.V., and Greiner, D.L.(2012). Humanized mice for immune system investigation: progress,promise and challenges. Nat Rev Immunol 12, 786-798.

Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P.,Eberl, G., Koyasu, S., Locksley, R.M., McKenzie, A.N., Mebius, R.E., etal. (2013). Innate lymphoid cells--a proposal for uniform nomenclature.Nat Rev Immunol 13, 145-149.

Vély, F., Barlogis, V., Vallentin, B., Neven, B., Piperoglou, C., Ebbo,M., Perchet, T., Petit, M., Yessaad, N., Touzot, F., et al. (2016).Evidence of innate lymphoid cell redundancy in humans. Nat Immunol 17,1291-1299.

Yagi, R., Zhong, C., Northrup, D.L., Yu, F., Bouladoux, N., Spencer, S.,Hu, G., Barron, L., Sharma, S., Nakayama, T., et al. (2014). Thetranscription factor GATA3 is critical for the development of allIL-7Rα-expressing innate lymphoid cells. Immunity 40, 378-388.

Yang, Q., Li, F., Harly, C., Xing, S., Ye, L., Xia, X., Wang, H., Wang,X., Yu, S., Zhou, X., et al. (2015). TCF-1 upregulation identifies earlyinnate lymphoid progenitors in the bone marrow. Nat Immunol 16,1044-1050.

Zook, E.C., Ramirez, K., Guo, X., van der Voort, G., Sigvardsson, M.,Svensson, E.C., Fu, Y.X., and Kee, B.L. (2016). The ETS1 transcriptionfactor is required for the development and cytokine-induced expansion ofILC2. J Exp Med 213, 687-696.

1-15. (canceled)
 16. A purified population of innate lymphoid cellprecursors (ILCPs), wherein at least 75% of the cells in the populationhave the phenotype CD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+, CD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD62L+, orCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+CD62L+.
 17. The purifiedpopulation of ILCPs of claim 16, wherein at least 90% of the cells inthe population have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+, CD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD62L+, orCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+CD62L+.
 18. The purifiedpopulation of ILCPs of claim 16, wherein at least 75% of the cells inthe population have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+.
 19. The purified populationof ILCPs of claim 16, wherein at least 75% of the cells in thepopulation have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD62L+.
 20. The purified populationof ILCPs of claim 16, wherein at least 75% of the cells in thepopulation have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+CD62L+.
 21. The purifiedpopulation of ILCPs of claim 17, wherein at least 90% of the cells inthe population have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+.
 22. The purified populationof ILCPs of claim 17, wherein at least 90% of the cells in thepopulation have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD62L+.
 23. The purified populationof ILCPs of claim 17, wherein at least 90% of the cells in thepopulation have the phenotypeCD127+CD117+CD3-CRTh2-CD7+CD94-NKp44-CD26+CD62L+.
 24. The purifiedpopulation of ILCPs of claim 21, comprising at least 10⁴ ILCPs cells.25. The purified population of ILCPs of claim 22, comprising at least10⁴ ILCPs cells.
 26. The purified population of ILCPs of claim 23,comprising at least 10⁴ ILCPs cells.
 27. The purified population ofILCPs of claim 21, comprising at least 10⁶ ILCPs cells.
 28. The purifiedpopulation of ILCPs of claim 22, comprising at least 10⁶ ILCPs cells.29. The purified population of ILCPs of claim 23, comprising at least10⁶ ILCPs cells.
 30. The purified population of ILCPs of claim 24, in amedium containing IL-2 and IL-1β.
 31. The purified population of ILCPsof claim 25, in a medium containing IL-2 and IL-1β.
 32. The purifiedpopulation of ILCPs of claim 26, in a medium containing L-2 and IL-1β.33. The purified population of ILCPs of claim 27, in a medium containingIL-2 and IL-1β.
 34. The purified population of ILCPs of claim 28, in amedium containing IL-2 and IL-1β.
 35. The purified population of ILCPsof claim 29, in a medium containing IL-2 and IL-1β.